Un Grosso Grazie Alle Carrube

Alessio, Jacopone, Luca Volanti Massi

Isa Anche

Gianmà, Lorenz, Damiano, Nardin, Luca, Borto, Kaus Elena Taglia

ovviamente Bruzzone ) Ci Vediamo Presto Al Tolo

Alla Mia Famiglia: Iosella

Enrico Fabio

Ai Nonni Giuliana

Guglielmo Grazie

Jaume, chomechome, el mau… I reserved a special space for you! Thank you, my friend, I'll limit myself to few lines because I could write another thesis about our life at the Vivaraize. It

LIST OF ABBREVIATIONS

maintenu pendant ces trois années a été la clé pour mener à bien ce travail de thèse. On a eu des difficultés pendant la thèse (une pandémie mondiale, pour en citer une), mais je pense que nous avons fait du mieux que nous pouvions et qu'on s'en est même très bien sorti.

Vianney, désolé de ne pouvoir partager ta passion pour le foot, je n'y comprends rien ! Même en sports de raquette je suis nul, je sais qu'au paddle tu voulais griller au moins Thomas, mais t'es tombé sur un mec que ne sait que courir et faire du snow. Merci d'avoir supporté mon projet de formation de moniteur de snowboard : si jamais l'arbitrage ne t'apporte pas assez d'adrénaline, ou si tu n'as jamais visité les urgences pour un poignet cassé, t'as mon 06. T'as été le directeur de thèse dont j'avais besoin, même si parfois les gens nous échangent le rôle (tu pourrais te faire pousser quelque cheveu blanc !).

Thomas, au début tu me faisais peur, car tu utilises des expressions compliquées parfois et t'as une humeur bizarre, mais mon français s'est amélioré et hier j'ai même réussi à te faire un clash (j'avoue que j'ai eu une décharge d'adrénaline quand même). Merci pour nos temps d'échange, la mise à disposition de ton expérience et pour les projets dont tu m'as investi.

Léo, merci pour ta gentillesse et ton aide sur les parties un peu plus cliniques-biologiques de ma thèse.

Et bien sûr, pour ton support à mes activités hors travail (ndr. rien de louche les gens, on parle de course à pieds dans le Pilat ici) sur Strava. On se verra l'année prochaine sur la ligne de départ à Tiranges peut être ! Merci à mon premier coloc, Gui. Merci de m'avoir accueilli au Canada, de m'avoir envoyé l'offre de thèse qui a changé ma vie, de m'avoir appris la science de la fatigue et de m'avoir fait redécouvrir le ski de fond que j'avais laissé de côté pendant 10 ans (je te promets que maintenant la technique est un peu amélioré ! on se reverra à la Marcialonga). Merci de m'avoir investi dans des projets de performance que feront toujours partie de mon bagage d'expérience. Je tiens aussi à te remercier pour ton travail en tant que directeur du LIBM, on a rarement la chance de travailler dans un si beau labo Un grand merci à Eric pour ton aide, ton amitié et disponibilité pour les manips (et pour les appels aux personnes âgées) ! J'espère que cette expérience pourra te servir pour la thèse aussi ! Bonne chance et courage ! X Je ne pourrai jamais remercier assez toutes les personnes qui ont donné leur temps et efforts pour participer aux études, et merci aussi a Clarence et Nathan qui m'ont apporté une aide précieuse pour la deuxième manip de cette thèse.

Un remerciement spécial a la bande du bureau C104. Claire, pour l'amitié et le chocolat, j'adore le chocolat ! Super merci ! Marie, mariafrangetta, le ninja heureusement que mon coeur est en pleine forme, avec toutes les peurs que tu me fais prendre. Mais j'ai pris ma revanche… une fois. On est à combien, 45-1 ? Merci pour le support en fin thèse, pour le temps à venir, embête les autres avec les bulles de neige de ma part aussi. Loic, el Loico ardechois, Mr. Pfffff, el mas guapo del barrio, on en a dit des bêtises ces années. Tu me rends fier quand j'entends t'appeler « quiqueeeee » dans le couloir.

Désormais c'est parti pour les grand courses, on se verra un jour à l'UTMB j'espère ! Thibault, Tibolo, 42man, le keyboarder… ça y est ! C'est fait, on est (presque) à la fin. J'aurai tellement nostalgie de toi, à qui je dirai chaque truc qui me passe par l'esprit ? Qui il y aura dernier mon écran la prochaine fois ? Qui sera le sage du laboratoire avec la plus grande responsabilité ?

Merci à tous pour ces années inoubliables. Anders, Fuglenskiderpataget, tu seras le probable héritier de ma place au C104, occupe-la avec honneur, Jaume va t'apprendre comment faire, bonne chance.

To my friends, los tiburones de Saint-Etienne. José, the handsome one with the fiat 500XXL, the man that can speak every language, thank you for making me addict to the pasteis de nata. Callum, mate, remember that for making coffee usually you put more water than powder… good for deadlines though! Hope to come visit you in Scotland soon. Fredo, oh Fredo el T-rex que pasa chamaco? please buy a new cycling jersey soon, a stylish one. Style is important, so take out this bolsa from the seat of your poor bike. And stop arguing with Quique, you know that lions are not dangerous! And… We still have this unfinished business at the Valencia Marathon. Let's do it! Quique… mixed model?

Where??? Stop speaking about stats! But I like when you try to explain me some models (what was the last one? Beta-regressions?), you are so happy! Looking forward for your papiro in Padova.

INTRODUCTION (FRANÇAIS)

D'ici 2050, l'espérance de vie mondiale à la naissance devrait passer d'environ 70 ans à 76 ans, la population âgée de plus de 65 ans devrait passer de ~9% de la population à ~17%, et la partie la plus âgée de la population (>80 ans) pourrait tripler, passant de 127 à 447 millions de personnes. Comme le vieillissement s'accompagne d'une augmentation du taux d'hospitalisation et des coûts des soins de santé, les conséquences d'une augmentation de la population âgée et très âgée sur le budget sanitaire seraient un lourd fardeau. Les altérations physiologiques liées au vieillissement ont montré un intérêt grandissant de la part de la communauté scientifique, afin de comprendre et d'atténuer ces détériorations. Les connaissances actuelles sur les processus du vieillissement mettent en évidence un déclin physiologique continu chez les individus en bonne santé, entraînant une limitation de la capacité à accomplir les tâches de la vie quotidienne (par exemple marcher, retrouver l'équilibre, porter des poids).

Ce déclin physiologique lié à l'âge, en l'absence de maladie particulière, commence approximativement à 30 ans. Chaque décennie, la consommation maximale d'oxygène diminue d'environ 10 % [START_REF] Trappe | Aging among elite distance runners: a 22-yr longitudinal study[END_REF][START_REF] Baker | Aging Performance for Masters Records in Athletics, Swimming, Rowing, Cycling, Triathlon, and Weightlifting[END_REF], la fréquence cardiaque maximale d'environ 7 bpm [START_REF] Tanaka | Age-predicted maximal heart rate revisited[END_REF], et la masse musculaire d'environ 5% (Mitchell et al. 2012). Alors que des études sur des athlètes Master ont démontré qu'il était possible de maintenir une performance athlétique relativement bonne jusqu'à 50 ans ou plus [START_REF] Baker | Percentage decline in masters superathlete track and field performance with aging[END_REF][START_REF] Knechtle | Age-related changes in 100-km ultramarathon running performance[END_REF], un déclin rapide est inévitablement observé à partir de la 7e décennie, tant en termes de performance que de fonction du système neuromusculaire [START_REF] Faulkner | Age-related changes in the structure and function of skeletal muscles[END_REF]). De plus, ce processus semble s'exacerber au cours de la 9e décennie. Par exemple, la perte de masse musculaire s'accélère, passant de ~0,5 % à ~1 % par an, tandis que la perte de force et de puissance est encore plus importante, de l'ordre de -3 % chaque année [START_REF] Skelton | Strength, power and related functional ability of healthy people aged 65-89 years[END_REF]Mitchell et al. 2012). Ce déclin soudain de la qualité musculaire chez les octogénaires comprend des modifications de la taille, de la forme, de l'architecture, de la vascularisation, de la morphologie des fibres et de la synthèse des protéines, accompagnées d'une réduction des concentrations de myonucléus et de cellules satellites, et d'une augmentation de la graisse intramusculaire et du stress oxydatif (Mitchell et al. 2012). De plus, cette détérioration ne se limite pas au muscle, car les facteurs centraux (la partie du système neuromusculaire qui s'étend du cortex moteur à la jonction neuromusculaire) sont également influencés par le processus de vieillissement [START_REF] Clark | Weaker seniors exhibit motor cortex hypoexcitability and impairments in voluntary activation[END_REF]Sundberg et al. 2018b ;Rozand et al. 2020). Ainsi, l'étude des déficiences du système neuromusculaire qui surviennent avec le vieillissement est cruciale pour comprendre et prévenir les risques de fragilité, de perte d'autonomie et d'hospitalisations [START_REF] Hunter | The relevance of sex differences in performance fatigability[END_REF](Hunter , 2018)). La fonction du système neuromusculaire peut être divisée en quatre composantes principales : force, puissance, endurance et fatigabilité.

Alors que les altérations liées à l'âge de la capacité à générer et à maintenir la force ont été largement étudiées et semblent être dues à une perte progressive de la masse musculaire et du tissu neural, qui dépend de leur utilisation (Power et al. 2016b), la fatigabilité est plus complexe et n'a pas été étudiée en profondeur dans la population très âgée. Par exemple, les altérations de la fonction neuromusculaire liées à l'âge pourraient varier en fonction des différentes tâches/activités effectuées (Hunter 2018). Une revue récente (Krüger et al. 2018) a démontré que, lorsque la charge de travail était normalisée par la capacité maximale individuelle, les personnes âgées étaient moins fatigables après des exercices isométriques, c'est-à-dire qu'ils présentaient une perte de la force maximale isométrique moins importante. Lorsque l'exercice était dynamique, les personnes âgées démontraient une fatigabilité similaire à celle de leurs homologues plus jeunes. Bien que la normalisation de la charge de travail par les capacités maximales est une approche intéressante pour étudier les propriétés du système neuromusculaire, son application fonctionnelle reste limitée (Hureau et al. 2021). Régler la charge en pourcentage du poids de corps pourrait apporter des connaissances transférables aux activités de la vie quotidienne. En effet, les activités comme la marche ou monter des escaliers dépendent à la fois de charges relative (par exemple le poids du corps) et de charges absolues (par exemple soulever une charge) (Hureau et al. 2021).

Pour ajouter de la complexité, le niveau d'activité physique quotidien pourrait jouer un rôle clé dans le maintien de la bonne fonctionnalité du système neuromusculaire avec le vieillissement.

Récemment, nous avons mis en lumière comment le comportement en matière d'activité physique peut être un facteur discriminant chez les personnes âgées (Varesco et al. 2021a) et, s'il n'est pas pris en compte, il pourrait conduire à des interprétations erronées des résultats. En évaluant des individus physiquement actifs, il serait possible d'évaluer les effets délétères du vieillissement uniquement plutôt que ceux du vieillissement combinés à l'inactivité physique (Harridge et Lazarus 2017).

Le but de cette thèse était d'investiguer la fonction neuromusculaire et la fatigabilité de personnes jeunes (<35 ans), âgées (>60 ans) et très âgées (>80 ans) lors de tâches isométriques et dynamiques avec différents types de charge. Seuls les individus actifs étaient intégrés aux études pour limiter les effets de l'inactivité physique.

INTRODUCTION (ENGLISH)

By 2050 the global life expectancy at birth is projected to increase from the actual ~70 years to ~76 years, the population over 65 years is expected to grow from ~9% of the population to ~17%, and the oldest part of the population (>80 years) could triplicate, from 127 to 447 million people. Because hospitalization rate and health care costs increase with age, the consequences of an increase in the old and very old populations on the sanitary budget would be a heavy burden. The age-related physiological alterations have shown increasing interest in the scientific community to understand and attenuate these deteriorations. Current understandings of the aging process evidence a continuous physiological decline in healthy individuals, leading to limitations in the ability to perform the tasks of the everyday life (e.g. walking, regain balance, carrying weights).

This age-related physiological decline, in absence of particular disease, starts approximatively at 30 years old. Each decade, maximal oxygen consumption declines by ~10% [START_REF] Trappe | Aging among elite distance runners: a 22-yr longitudinal study[END_REF][START_REF] Baker | Aging Performance for Masters Records in Athletics, Swimming, Rowing, Cycling, Triathlon, and Weightlifting[END_REF], maximal heart rate decreases by ~7 bpm [START_REF] Tanaka | Age-predicted maximal heart rate revisited[END_REF], and muscle mass by ~5% (Mitchell et al. 2012). While studies on master athletes demonstrated that maintaining a relatively good athletic performance is possible until 50 years old or more [START_REF] Baker | Percentage decline in masters superathlete track and field performance with aging[END_REF][START_REF] Knechtle | Age-related changes in 100-km ultramarathon running performance[END_REF], a rapid decline is inevitably observed starting about the 7 th decade of human life, for both performance and neuromuscular function [START_REF] Faulkner | Age-related changes in the structure and function of skeletal muscles[END_REF]). Furthermore, this process seems exacerbated in the 9 th decade. For instance, muscle mass loss accelerates from ~0.5% to ~1% per year, while force and power loss are even greater, approximatively -3% every year [START_REF] Skelton | Strength, power and related functional ability of healthy people aged 65-89 years[END_REF]Mitchell et al., 2012). This sudden decline in muscle capacity in octogenarians includes changes in muscle size, shape, architecture, vascularization, fibers morphology and protein synthesis, accompanied by a reduction in the concentrations of myonuclei and satellite cells, and an increase in intramuscular fat and oxidative stress (Mitchell et al. 2012).

Moreover, this deterioration is not limited to the muscle, as central factors (i.e. the part of the neuromuscular system that extends from the motor cortex to the neuromuscular junction) are also influenced by the aging process [START_REF] Clark | Weaker seniors exhibit motor cortex hypoexcitability and impairments in voluntary activation[END_REF]Sundberg et al. 2018b;Rozand et al. 2020). Thus, studying the impairments in the neuromuscular system that occur with aging is crucial to understand and prevent the risks of frailty, autonomy loss and hospitalizations (Newman et al. 2006;[START_REF] Cruz-Jentoft | Sarcopenia: European consensus on definition and diagnosis[END_REF][START_REF] Hunter | The relevance of sex differences in performance fatigability[END_REF]Hunter , 2018)). The function of the neuromuscular system has been mainly investigated by measuring strength, power, endurance and fatigability [e.g. (Hunter et al. 2005;Justice et al. 2014)].

While the age-related alterations in the ability to generate and maintain force have been widely

studied and seem to be due to a progressive loss in muscle mass and neural tissue (Power et al. 2016b), fatigability is more complex and has not been extensively studied in the very old population (> 80 years old). For instance, the age-related impairments in neuromuscular function could vary among tasks/activities (Hunter 2018). A recent review (Krüger et al. 2018) evidenced that, when the load was normalized to the individual maximal capacity, older adults were less fatigable after isometric exercises, i.e. lower decrease in maximal isometric force. When dynamic exercise was proposed, old individuals showed similar fatigability compared to young adults. Although workload normalization to maximal capacity is a useful approach to investigate the properties of the neuromuscular system, its functional application is limited (Hureau et al. 2021). Setting the workload as percentage of body weight could provide knowledge transferable to the activities of daily living. Indeed, actions like walking or climbing stairs depend on both relative (e.g. body weight) and absolute loads (e.g. carrying weights) (Hureau et al. 2021).

To further add complexity, the level of daily physical activity could play a key role in the maintenance of good functionality of the neuromuscular system with aging. Recently, we highlighted how physical activity behavior can be a discriminant factor among older adults (Varesco et al. 2021b) and could lead to misinterpretations of the results in absence of assessment. By evaluating physically active individuals it would be possible to assess the deleterious effects of inherent aging rather than aging in combination with physical inactivity (Harridge and Lazarus 2017).

The aim of the present thesis was to investigate neuromuscular function and performance fatigability in young (< 35 years), old (> 60 years) and very old (> 80 years) adults after isometric and dynamic fatiguing tasks with different workloads. Only active adults were considered to limit the effects of physical inactivity.

THEORETICAL FRAMEWORK

1. NEUROMUSCULAR FUNCTION AND FATIGABILITY

Fatigue and fatigability

Fatigue is a multifactorial and complex phenomenon that generally occurs following a stressorinduced change in the homeostasis of the organism. However, despite the large and evolving literature in the field of exercise physiology, a clear and complete definition of fatigue is still hard to find. In materials engineering, fatigue describes a structural failure that occurs following repeated cyclic loads, even when the experienced stress range is far below the static material strength (Britannica Online Encyclopedia 2021). This concept could be extended to exercise physiology, attributing 3 main characteristics to fatigue [START_REF] Craig | Human Muscle Fatigue[END_REF]: i) an alteration in the homeostasis of one or more biological systems, ii) this alteration is reversible with recovery and iii) it can happen before (latent fatigue) an observable decrease in the performance (i.e. exhaustion). Since the pioneer works of the Italian physiologist Angelo Mosso , it has been acknowledged that exerciseinduced fatigue is a complex phenomenon that encompass impairments from the central nervous system [CNS, [START_REF] Enoka | Unraveling the neurophysiology of muscle fatigue[END_REF]], down to the skeletal muscle level. This results in both the sensation of tiredness and the decline in muscle force and performance [START_REF] Enoka | Unraveling the neurophysiology of muscle fatigue[END_REF]Taylor et al. 2016a;[START_REF] Enoka | Translating fatigue to human performance[END_REF]. To separate those two distinct phenomenon, in recent years some authors suggested that the term "fatigue" should be separated in "perceived fatigability" and "performance fatigability" [START_REF] Enoka | Translating fatigue to human performance[END_REF]. "Perceived fatigability" is usually adopted to indicate the sensation of discomfort and lack of energy caused by exercise or clinical conditions [START_REF] Twomey | La fatigue neuromusculaire au cours de l'exercice : considérations méthodologiques, étiologie et rôle potentiel dans la fatigue chronique[END_REF]) and generally measured through visual scales of perceived effort/fatigue that answer to general questions like "how do you feel?" [START_REF] Micklewright | Development and Validity of the Rating-of-Fatigue Scale[END_REF][START_REF] St Clair Gibson | The interaction of psychological and physiological homeostatic drives and role of general control principles in the regulation of physiological systems, exercise and the fatigue process-The Integrative Governor theory[END_REF]. "Performance fatigability" indicates a decrease in observable performance (e.g. force, power)

following exercise [START_REF] Enoka | Translating fatigue to human performance[END_REF] and is usually quantified using dynamometers.

Performance fatigability and its etiology are typically quantified by evaluating impairments in the neuromuscular system (Figure 1), which is composed by the motor cortex (supraspinal or cortical component), the spinal motoneuronal pool (spinal component), and the motor units down to the actinmyosin bridges (peripheral or muscular component; Figure 1). A current definition of performance fatigability could be "a response that is less than the expected or anticipated contractile response, for a given stimulation" [START_REF] Macintosh | What is fatigue?[END_REF]. Usually, during exercise, afferent feedbacks stressed by exertion converge to the CNS, which could modulate the motor drive output to the muscles. This can be achieved by influencing the excitability/inhibition of three sites: the motor cortex, the motoneuron pool in the spinal cord pathway and/or the single motor units [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF][START_REF] Ranieri | The role of motor neuron drive in muscle fatigue[END_REF][START_REF] Heckman | Motor unit[END_REF]. Thus, performance fatigability could be caused by a lack in energetic substrates, oxygen supply, metabolite accumulation or structural alteration that happen within the muscle (impairments in contractile function), but also by alterations in the voluntary activation capacity of the skeletal muscle by the CNS (Figure 1).

Central origin of performance fatigability

The central origin of performance fatigability is multifactorial and depends on the population considered, the environmental conditions and the kind of task performed. From a practical point of view, it indicates the inability of the CNS to recruit the α-motoneurons at a frequency sufficient to provoke a tetanus during maximal contractions [START_REF] Millet | Electrical stimulation for testing neuromuscular function: From sport to pathology[END_REF]. Central alterations are usually defined as a decrease in voluntary activation of the muscle during exercise by processes not attributable to mental fatigue [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF]).

Biological processes

Different biological processes have been hypothesized to cause fatigue-related alterations within the CNS. The first phenomenon has been observed during prolonged exercise and involves the synthesis and metabolism of some neurotransmitters of the CNS such as serotonin, dopamine and noradrenalin that influence the activity of the serotonergic system [START_REF] Meeusen | Central fatigue: The serotonin hypothesis and beyond[END_REF]. Activity of the serotonergic system increases during fatiguing endurance exercise, causing sleepiness and general sensation of tiredness, and reduces the excitability (and thus the recruitment) of the motor unit pool [START_REF] Newsholme | Amino acids, brain neurotransmitters and a functional link between muscle and brain that is important in sustained exercise[END_REF][START_REF] Newsholme | The plasma level of some amino acids and physical and mental fatigue[END_REF]. An increase in the serotonergic system activity has also been shown to impair motor coordination, proprioception and the ability to focus, and to increase the reaction time to an external stimulus [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF]. Moreover, a decrease in the dopamine/serotonin ratio is well correlated with the decrease in maximal muscular voluntary activation [START_REF] Meeusen | Brain neurotransmitters in fatigue and overtraining[END_REF].

Other biochemical processes have been proposed to contribute to the development of voluntary activation impairments, such as the influence of several neurotransmitters [GABA, ACTH, glutamate and adenosine; [START_REF] Meeusen | Central fatigue: The serotonin hypothesis and beyond[END_REF]] that could be released in the CNS during exercise. These neurotransmitters can i) influence the neuronal excitation/inhibition within the motor cortex (GABA, glutamate), ii) reduce the secretion of dopamine and noradrenaline (adenosine) or iii) influence the energetic balance of the organism (ACTH). Other possible contributors to the alteration of the nervous system include i) the production of interleukins during exercise that provide an hormonal-like feedback [START_REF] Nybo | Cerebral perturbations provoked by prolonged exercise[END_REF][START_REF] Zając | Central and Peripheral Fatigue During Resistance Exercise -A Critical Review[END_REF], ii) an increased concentration of ammonium ions in the brain blood flow and iii) a reduction in the energetic precursors and oxygen to sustain the brain and muscle activity [START_REF] Nybo | Cerebral perturbations provoked by prolonged exercise[END_REF][START_REF] Meeusen | Central fatigue: The serotonin hypothesis and beyond[END_REF]).

Neural feedback from the muscles

The capacity to generate and transmit the voluntary drive to the muscles could be directly influenced by a feedback neural circuitry, i.e. inhibitory neurons that are activated by high concentrations of metabolic byproducts generated during exercise. This function has been mainly attributed to the groups III and IV afferent inputs [START_REF] Gandevia | Spinal and supraspinal factors in human muscle fatigue[END_REF][START_REF] Zając | Central and Peripheral Fatigue During Resistance Exercise -A Critical Review[END_REF][START_REF] Amann | Autonomic responses to exercise: group III/IV muscle afferents and fatigue[END_REF], which consist in myelinated and unmyelinated neural afferents to the CNS whose role is primarily to adjust the hearth rate, respiratory frequency and others hemodynamics factors during exercise [START_REF] Amann | Autonomic responses to exercise: group III/IV muscle afferents and fatigue[END_REF]. In this view, fatigability at CNS level would be considered as secondary to peripheral alterations [START_REF] Amann | Implications of group III and IV muscle afferents for high-intensity endurance exercise performance in humans[END_REF]. Afferent inhibitory burst within the spinal circuitry has been recognized as the main responsible for the reduction in spinal transmission. Thus, the motor drive to the α-motoneurons and then to the muscle can be reduced importantly at spinal level. Finally, intrinsic changes within the motoneurons and interneurons due to repetitive activation could also be responsible for spinal fatigability [START_REF] Kernell | Motoneurone properties and motor fatigue -An intracellular study of gastrocnemius motoneurones of the cat[END_REF][START_REF] Kukulka | Changes in human α-motoneuron excitability during sustained maximum isometric contractions[END_REF]). For instance, repetitive neuromodulator activation induces a dendritic persistent inward current that may increase the gain of motor excitability from 6 to 10 fold [START_REF] Heckman | Persistent inward currents in spinal motoneurons and their influence on human motoneuron firing patterns[END_REF]). This phenomenon is mediated by the CNS, i.e. noradrenergic neurons (from the locus coeruleus) and serotoninergic neurons (from the caudal raphe nucleus) send their axons down to the spinal motoneurons. Serotonin release may increase α-motoneuron excitability when acting on some specific receptors while high concentrations of serotonin leads to reduction in responsiveness of the targeted α-motoneuron [START_REF] Cotel | Serotonin spillover onto the axon initial segment of motoneurons induces central fatigue by inhibiting action potential initiation[END_REF]Taylor et al. 2016b). As serotonin release in those areas might decrease with exercise, this would result in a progressive decrease in spinal loop excitability during long lasting fatiguing tasks [START_REF] Kavanagh | Enhanced availability of serotonin increases activation of unfatigued muscle but exacerbates central fatigue during prolonged sustained contractions[END_REF]).

Peripheral origin of performance fatigability

The term "peripheral" denotes all the alterations that occur below the neuromuscular junction and that are associated with a loss in functionality of the contractile apparatus (e.g. diminution of sarcolemmal excitability, impairments in the actin-myosin coupling, metabolites accumulation and acute structural damage). The mechanisms that could be impaired are dependent on the type, intensity and duration of the task. The cascade of mechanisms that goes from the electrical signal to the neuromuscular junction to the force generation are presented in Figure 2. Two main categories of phenomena are accounted for this loss in contractile function: ionic and metabolic impairments.

Ionic mechanism

Ionic phenomena linked to the synaptic and membrane transmission of the action potential have a large safety factor. This means that the concentration of ions displaced from the activity of the Na + /K + pump and the displacement of Cl -ions, largely overcome the quantity needed for an overshoot (action potential). Then, depletion is unlikely and this mechanism probably does not limit exercise performance (Allen et al. 2008b, a). However, exceptions occur in case of transient ions accumulations in small volumes [START_REF] Sejersted | Dynamics and consequences of potassium shifts in skeletal muscle and heart during exercise[END_REF]. This is the case for the action potential transmission across the T-tubules, because of their very small volume [less than 1% of the muscle; [START_REF] Farrell | ACSM's Advanced Exercise Physiology[END_REF]]. During the repeated activity of the Na + /K + pump, there is an extracellular accumulation of K + ions and depletion of Na + ions. Those transitory events increase in magnitude with high discharge frequencies, inactivating the Na + channels and reducing the action potential transmission. Consequently, there is a lower activation of the voltage-dependent ryanodine receptors, and in turn a reduced release of Ca 2+ from the sarcoplasmic reticulum (SR) (MacLaren and Morton

2012).

Nevertheless, these localized ionic phenomena in vivo have been considered secondary (Allen et al. 2008b). Indeed, other ionic mechanism have been hypothesized to compensate for the K + accumulation, such as a reduction in the conductance of the ions Cl -caused by the drop in pH during exercise [START_REF] Pedersen | Increased Excitability of Acidified Skeletal Muscle[END_REF]. Furthermore, other alterations in contractile function occur before the accumulation of ions in the T-tubules, and could play an important role in Ca 2+ release (Allen et al. 2008a;[START_REF] Maclaren | Biochemistry for sport and exercise metabolism[END_REF]. One of those involves the local depletion of ATP in the interspace between T-tubules and SR [START_REF] Stewart | Protection of muscle membrane excitability during prolonged cycle exercise with glucose supplementation[END_REF]. In this interspace, the high concentration of glycolytic enzymes can be unable to produce ATP in case of lack in substrate supply (Ørtenblad et al. 2011). This ATP is necessary for the functioning of the ATP-dependent channels like the Na + /K + pumps or the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), which are responsible for the calcium reuptake (Ørtenblad et al. 2011). Reduction in calcium reuptake has been associated with the slowdown in the rate of muscle relaxation observed with fatigue [START_REF] Vernillo | Use of transcranial magnetic stimulation to assess relaxation rates in unfatigued and fatigued knee -extensor muscles[END_REF].

Other ionic mechanisms of fatigability consists in ionic imbalance. Magnesium (Mg 2+ ) depletion favours an excessive quantity of Ca 2+ to binds the troponin C, resulting in hypercontractility and muscular cramps/spasm [START_REF] De Baaij | Magnesium in Man: Implications for Health and Disease[END_REF]. In case of ATP depletion, there is a rise in free Mg 2+ [START_REF] Westerblad | Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle[END_REF]. Accumulation of Mg 2+ could also have deleterious effects on SERCA and ryanodine receptors, and thus on Ca 2+ release and reuptake [START_REF] Blazev | Low [ATP] and elevated [Mg2+] reduce depolarization-induced Ca2+ release in rat skinned skeletal muscle fibres[END_REF][START_REF] Dias | Insights into the Regulation of the Ryanodine Receptor: Differential Effects of Mg 2+ and Ca 2+ on ATP Binding[END_REF][START_REF] De Baaij | Magnesium in Man: Implications for Health and Disease[END_REF].

Metabolic mechanisms: the role of pH and free phosphates

Classically, a decrease in oxygen uptake from the blood flow has been linked to the utilisation of the lactic anaerobic system and accounted as responsible for the production of lactate (La -) and H + ions.

H + ions accumulation decreases cellular pH, inducing alterations in contractile function. In particular, the drop in pH seems associated with the slowing of the rate of force relaxation [START_REF] Westerblad | Recent advances in the understanding of skeletal muscle fatigue[END_REF]. However, several studies showed that the drop in pH and the presence of lactate would not be determinant for other contractile function impairments (Stackhouse et al. 2001a;[START_REF] Westerblad | Recent advances in the understanding of skeletal muscle fatigue[END_REF]Allen et al. 2008b;[START_REF] Maclaren | Biochemistry for sport and exercise metabolism[END_REF].

In the last few decades, research focusing on the metabolic causes of peripheral origin of neuromuscular fatigue found a key role played by the free phosphate (Pi) released following exercise, as it regulates several biochemical pathways (Baker et al. 2010). Accumulation of Pi has been shown

to reduce or even directly inhibit the formation of actin-myosin bridges and bind the calcium released from the SR, blocking its affinity with the troponin C and SERCA [START_REF] Dahlstedt | Role of myoplasmic phosphate in contractile function of skeletal muscle: studies on creatine kinase-deficient mice[END_REF][START_REF] Westerblad | Recent advances in the understanding of skeletal muscle fatigue[END_REF]Allen et al. 2008b;[START_REF] Debold | Recent insights into the molecular basis of muscular fatigue[END_REF]. Concomitant with the increase in Pi production, high energetic turnover causes the depletion of glucose and glycogen. Impairments in glucose or energetic supply to the muscle leads to ATP depletion and the accumulation of ADP and AMP [START_REF] Cooke | The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate[END_REF]Allen et al. 2008b). ADP and AMP are important allosteric effectors involved in cell and glucose metabolisms, regulating the energetic production and consumption [START_REF] Jensen | AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: Necessary and/or sufficient[END_REF].

Concentration of free radicals

Prolonged energetic turnover during exercise causes the rise in concentration of by-products such as free radical species, i.e. the reactive oxygen species (ROS) and reactive nitrogen species (RNS) [START_REF] Reid | Free radicals and muscle fatigue: Of ROS, canaries, and the IOC[END_REF][START_REF] Ferreira | Muscle-derived ROS and thiol regulation in muscle fatigue[END_REF], which could damage the cell structure. The production of free radicals could increase dramatically during intense and prolonged exercises as well as in ischemic conditions, dehydration, UV radiation exposure, heat, infections, lack of sleep or poor diets [START_REF] Lewis | Alterations in Redox Homeostasis in the Elite Endurance Athlete[END_REF].

ROS/RNS accumulation causes muscle structural damage at the muscular proteins level, in particular to troponin C, myosin and actin, reducing in turn Ca 2+ sensibility and contractile force [START_REF] Scherer | Oxidative stress impairs the function of sarcoplasmic reticulum by oxidation of sulfhydryl groups in the Ca2+-ATPase[END_REF][START_REF] Snook | Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility[END_REF][START_REF] Debold | Recent insights into the molecular basis of muscular fatigue[END_REF]). An association between those free radical species and performance fatigability has been directly observed by experimentally exposing muscle fibers to ROS-scavengers, which reduced the concentration of free radicals within the cell. As a result, force depression was attenuated (Moopanar andAllen 2005, 2006).

TAKE HOME MESSAGE: NEUROMUSCULAR FUNCTION AND FATIGABILITY

Fatigue can be separated in two main aspects: perceived fatigability and performance fatigability.

Performance fatigability originates within the neuromuscular system. Performance is influenced by the intensity/workload of exercise. To compare performance between different age populations, a typical approach in the laboratory setting is to normalize intensity to maximal capacity of each individual, e.g. percentage of the maximal voluntary contraction force or power (Krüger et al. 2018), and performing single-limb fatiguing tasks.

Performance fatigability

The standard approach for quantifying performance fatigability is measuring directly the difference in maximal force or power before and after a fatiguing task [START_REF] Vøllestad | Measurement of human muscle fatigue[END_REF]. In aging studies, the most diffused approach to quantify performance fatigability is to measure the loss in maximal isometric force (Krüger et al. 2018). The alterations in neuromuscular function varies if performance fatigability is quantified using single-limb isometric contractions (e.g. maximal isometric contractions) or single-limb (e.g. maximal concentric contraction) and multiarticular dynamic contractions (e.g. maximal sprints on bike ergometers) in both fresh and fatigued muscle (Krüger et al. 2018(Krüger et al. , 2019)). Despite most of the activities of daily living are performed using dynamic multiarticular contractions, monoarticular isometric contractions allow the experimental investigation of performance fatigability etiology using artificial stimulations (electrical or magnetic) techniques.

Indeed, it is technically challenging to perform neuromuscular evaluation during dynamic contractions, even if some attempt have been performed [e.g. [START_REF] Sidhu | Motor cortex excitability does not increase during sustained cycling exercise to volitional exhaustion[END_REF][START_REF] Weavil | Fatigue diminishes motoneuronal excitability during cycling exercise[END_REF]].

Performance fatigability etiology

The most used technique to evaluate the etiology of performance fatigability and quantify the presence of peripheral (contractile function) vs. central (voluntary activation) alterations is the interpolated twitch technique [ITT; [START_REF] Shield | Assessing Voluntary Muscle Activation with the Twitch Interpolation Technique[END_REF]]. Briefly, the interpolated twitch technique consists in evoking a response to artificial stimulation during a maximal isometric contraction, and another at rest after the contraction. These responses are then compared before and after a fatiguing task (for further details see section 2.3.3). Different techniques are available to study the etiology of performance fatigability: magnetic stimulation that could be applied over the motor cortex or the peripheral nerve, or electrical stimulation that could be applied on the peripheral nerve or directly over the muscle belly. The frequency and intensity of stimulation can also be modulated.

The parameters are selected depending on the population and the outcome studied. For instance, highfrequency doublets could better detect impairments in voluntary activation (Rozand et al. 2020) but the discomfort related to those procedures induce some research groups to use single stimulations when studying specific populations such as the old adults or patients [START_REF] Millet | Potential interests and limits of magnetic and electrical stimulation techniques to assess neuromuscular fatigue[END_REF]Bachasson et al. 2013).

Measurement of peripheral alterations

Contractile function can be measured analyzing the responses evoked at rest by artificial electrical or magnetic stimulation over the motor nerve or the muscle belly. Stimulation techniques allow to quantify peripheral neuromuscular alterations. However, it is not possible to assess directly the biological mechanisms that could be responsible for muscle fatigability with these techniques.

However, biological techniques such as biopsies or blood sample require sampling biological tissues, which is invasive and usually limited to a small and superficial muscle compartment. The information are usually specific to the muscle and do not allow the investigation of the whole neuromuscular system including the central nervous system. Although stimulation being an indirect technique, it offers a vast possibility of application, from "on field" studies [e.g. [START_REF] Besson | Sex Differences in Neuromuscular Fatigue and Changes in Cost of Running after Mountain Trail Races of Various Distances[END_REF]] to clinical populations [e.g. [START_REF] Souron | Relationship between intensive care unit-acquired weakness, fatigability and fatigue: What role for the central nervous system[END_REF]]. Originally, high-frequency trains of peripheral nerve stimulations (PNS) evoked at rest were used to measure the maximal contraction capacity of the muscle measured on a dynamometer. With this procedure, it is possible to obtain a fused tetanus in the studied muscle. However, the discomfort of the evoked stimuli induced the researchers to progressively substitute high-frequency tetanus with high-frequency octet, doublets, or single twitches [START_REF] Millet | Electrical stimulation for testing neuromuscular function: From sport to pathology[END_REF]). The choice is usually based on the number of stimulations delivered during the protocol and the tolerance of the single individual or the population considered, other than the safety of the procedures. For instance, high-frequency stimulation trains induce risks of knee cap dislocation [START_REF] Bigland-Ritchie | Central and Peripheral Fatigue in Sustained Maximum Voluntary Contractions of Human Quadriceps Muscle[END_REF], limiting its use for aging studies. When evoking a response at rest (Figure 3), the intensity and duration of the stimulation are adjusted to recruit all the muscle mass available for contraction. Long impulses and high intensities of stimulation could help to maximally recruit large muscle masses such as the quadriceps [e.g. [START_REF] Gregory | Impact of varying pulse frequency and duration on muscle torque production and fatigue[END_REF][START_REF] Zarkou | Comparison of techniques to determine human skeletal muscle voluntary activation[END_REF]]. If EMG signal is recorded during single stimulations at rest, the maximal M-wave (Figure 3) obtained by increasing stimulation intensity corresponds to the simultaneous activation of all the motor units of the muscle [START_REF] Hugon | Methodology of the Hoffmann Reflex in Man[END_REF]. The parameters usually studied from the evoked response on the force channel (peak twitch, Figure 3) are its amplitude, contraction time and half-relaxation time. The comparison of the peak twitch amplitude prior and after a fatiguing task is adopted as global measure of the fatigue-induced impairments in contractile function. In several studies attempting to detect in vivo some age-related differences in calcium kinetic, the contraction time and half-relaxation time of the response evoked at rest by nerve stimulation has been used to quantify calcium release and reuptake, respectively (Krüger et al. 2018). However, the discordant results across studies, probably due to high between-subjects variability, and low sensibility of these parameters compared to the peak relaxation rate detected from the drop in force following a stimulation over the motor cortex [e.g. [START_REF] Vernillo | Use of transcranial magnetic stimulation to assess relaxation rates in unfatigued and fatigued knee -extensor muscles[END_REF]] lower their usefulness in aging studies (Krüger et al. 2018).

To evaluate possible impairments in calcium release in fresh muscle, two different doublets, as surrogates of similar-frequencies evoked tetani (100 Hz and 10 Hz), are delivered for the assessment of the low-to-high frequency fatigue [LFF, [START_REF] Verges | Comparison of electrical and magnetic stimulations to assess quadriceps muscle function[END_REF]]. The decrement in force at low frequency (10 Hz) compared to high frequency (100 Hz), in absence of muscle damage, is mainly attributed to a decrease in cytosolic free calcium concentration ([Ca 2+ ]i) due to a lower release in calcium from the SR, indicating muscle damage. Due to the possible partial compensation of postactivation potentiation (i.e. transient increase in calcium sensitivity following an intense contraction) to LFF [START_REF] Sale | Postactivation potentiation: Role in human performance[END_REF], timing, number and order of stimulation, and position and length of the studied muscle [START_REF] Kohn | Voluntary activation and twitch potentiation of the elbow flexors across supinated, neutral, and pronated forearm orientations[END_REF]) must be kept constant before and after the task [START_REF] Ruggiero | Maximal results with minimal stimuli : the fewest high-frequency pulses needed to measure or model prolonged lowfrequency force depression in the dorsiflexors[END_REF]. Pairing PNS and EMG signals, it is possible to measure the compound muscle action potential (CMAP, or M-wave, Figure 3), which represents the summated electrical activity of the motor units of the depolarized motor axons. The M-wave could provide useful information on the excitability of the sarcolemma, although it represents only the activity of the motor units within the recording range of the electrodes (Figure 3). Whether an increase or a decrease in the amplitude of the M-wave would indicate an impairment, however, is still under discussion (Rodriguez-Falces and Place 2018).

An alternative to electrical nerve stimulation is magnetic nerve stimulation [START_REF] Verges | Comparison of electrical and magnetic stimulations to assess quadriceps muscle function[END_REF]). The advantage offered by peripheral nerve magnetic stimulation (PNMS) consists in reducing the discomfort induced by electrical stimulation over the peripheral nerve, which can be relevant when stimulating large muscle masses such as the knee extensors in old and very old adults. Despite some evidence suggest its validity [e.g. [START_REF] Olney | A comparison of magnetic and electrical stimulation of peripheral nerves[END_REF][START_REF] Nilsson | Determining the site of stimulation during magnetic stimulation of a peripheral nerve[END_REF][START_REF] Neyroud | Comparison of electrical nerve stimulation, electrical muscle stimulation and magnetic nerve stimulation to assess the neuromuscular function of the plantar flexor muscles[END_REF]], this technique has not been widely adopted in aging studies because of uncertainty of the stimulation site and because the fat tissue could interfere with the efficacy of the stimulation (Tomazin et al. 2011).

Evoking high-frequency doublets evoked via PNMS could represent a possible solution because they are less painful than doublets elicited via electrical stimulations and the responses obtained are greater than that with single stimuli, possibly overcoming some of the limitations related to fat thickness (Tomazin et al. 2011). However, due to stimulation artifact contamination, the M-wave elicited via PNMS cannot be consistently recorded [START_REF] Neyroud | Comparison of electrical nerve stimulation, electrical muscle stimulation and magnetic nerve stimulation to assess the neuromuscular function of the plantar flexor muscles[END_REF]. Some efforts have been made to suppress artificially the artefact and synthetically reconstruct the contaminated signal [START_REF] Liu | Suppression of stimulus artifact contaminating electrically evoked electromyography[END_REF], however, peripheral nerve electrical stimulation remains the gold standard for the assessment of M-waves.

Measurement of cortical and spinal excitability and inhibition

Stimulation techniques allow to test the excitability and behaviour of the motoneurons along the neuromuscular pathway. Variation in excitability is defined as the variation in the recruitment of more or less motoneurons to generate an action potential during or following an intervention [START_REF] Mcneil | Testing the excitability of human motoneurons[END_REF]. This variation comes from the interaction of different mechanisms of inhibition and facilitation across the neuromuscular pathway [START_REF] Mcneil | Testing the excitability of human motoneurons[END_REF][START_REF] Amann | Autonomic responses to exercise: group III/IV muscle afferents and fatigue[END_REF]. In vivo, the proprieties of the motoneuronal pool in humans are necessarily measured indirectly. Impairments in the transmission of the signal from the motor cortex to the motor units have been assessed for long time through the Hoffmann reflex (H-reflex): a submaximal percutaneous electrical stimulation is delivered to activate selectively the neural afferences firing to the alpha-motoneuron. This allows the quantification of the spinal loop excitability [START_REF] Zehr | Considerations for use of the Hoffmann reflex in exercise studies[END_REF][START_REF] Brinkworth | Standardization of H-reflex analyses[END_REF]. More recently, other variables obtained by electrical or magnetic stimulation have been used to study the spinal and supraspinal changes in excitability/inhibition [START_REF] Zehr | Considerations for use of the Hoffmann reflex in exercise studies[END_REF][START_REF] Mcneil | Testing the excitability of human motoneurons[END_REF] and have been applied in aging studies [e.g. [START_REF] Hunter | Recovery from supraspinal fatigue is slowed in old adults after fatiguing maximal isometric contractions[END_REF][START_REF] Baudry | Influence of age and posture on spinal and corticospinal excitability[END_REF]]. Stimulation of the corticospinal tract at the cervicomedullary level, over the cervical spine, evokes the socalled cervicomedullary motor evoked potential (CMEP). When elicited at the thoracic level, the EMG response is denominated thoracic motor evoked potential (TMEP). CMEP and TMEP, used respectively to investigate the upper and lower limbs motoneuron excitability, have the advantage to be mainly monosynaptic and unaffected by presynaptic inhibition [START_REF] Taylor | Stimulation at the cervicomedullary junction in human subjects[END_REF]Rösler, Scheidegger, & Magistris, 2009;[START_REF] Mcneil | Testing the excitability of human motoneurons[END_REF]. It is also possible to stimulate the primary motor cortex in order to elicit a motor evoked potential (MEP), via transcranial magnetic stimulation (TMS), to study the corticospinal excitability (Figure 4). To separate the excitability of the supraspinal tract (cortical excitability) from the corticospinal tract, the MEP could be normalized to the CMEP (upper limbs) or TMEP (lower limbs).

Another useful index when investigating the effect of an acute fatiguing task, the chronic adaptations to a training program or the effects of a treatment is the intracortical inhibition: following a MEP elicited during voluntary contractions, there is a flat period in the EMG signal: the so-called cortical silent period (SP, Figure 4). From the beginning of the SP, the first part is attributed to spinal process (up to 100 ms), while the second part represents the intracortical inhibition mediated by long-lasting GABAB receptors [START_REF] Sidhu | Corticospinal Responses to Sustained Locomotor Exercises: Moving beyond Single-Joint Studies of Central Fatigue[END_REF][START_REF] Gruet | Stimulation of the motor cortex and corticospinal tract to assess human muscle fatigue[END_REF].

Voluntary activation

The evaluation of voluntary activation was initially developed by Merton (Merton 1954) who used motor nerve electrical stimulation to elicit a tetanus and compared it to a maximal voluntary isometric contraction (MVIC) of the abductor pollicis. Merton reported a similar force expressed by the tetanus and the maximal voluntary contraction, concluding that this muscle could be fully activated. Despite the technological limitations, he demonstrated that a linear negative relationship exists between the evoked twitch amplitude and voluntary isometric force, i.e. the lower the target force level (expressed as % of MVIC), the higher the evoked force. He then used a technique based on electrical stimulations to study the voluntary activation, which he defined as the difference between the force derived from the maximal level of voluntary drive produced by the CNS and the absolute maximal force that can be produced by the muscle.

Voluntary activation can be modulated or impaired potentially at every level of the neuromuscular pathway [START_REF] Meeusen | Fatigue: Is it all neurochemistry[END_REF], starting from the motor cortex excitability, inhibition mediated by GABAB receptors or maybe the effects of other neurotransmitter in the brain [START_REF] Meeusen | Fatigue: Is it all neurochemistry[END_REF]. It is possible to establish in a semi-quantitative-way the increasing influence of central origin of neuromuscular fatigue on performance by calculating the percentage of voluntary activation, using the ITT (Merton 1954): during a short MVIC (3-5 s), when the plateau is reached, a supramaximal stimulation is delivered to evoke a superimposed twitch (SIT). Another stimulation is delivered at rest with a minimal time delay from the MVIC to elicit a potentiated twitch (POT), to account for the postactivation potentiation [START_REF] Sale | Postactivation potentiation: Role in human performance[END_REF]. The amplitude of the SIT is then normalized to the amplitude of the POT. This normalization is necessary to account for alterations in the drive transmission within the muscle cells [START_REF] Shield | Assessing Voluntary Muscle Activation with the Twitch Interpolation Technique[END_REF].

Figure 5. Typical assessment of Voluntary Activation via peripheral nerve stimulation

Voluntary activation is usually calculated using the following formula:

𝑣𝑜𝑙𝑢𝑛𝑡𝑎𝑟𝑦 𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 (%) = [1 -( 𝑆𝐼𝑇 𝑃𝑂𝑇 )] * 100 Equation 1
A sub-maximal voluntary activation would indicate that some motor units are not firing fast enough to produce a fused tetanus or are not recruited at all. Consequently, it can be inferred that some modulation in the motor drive prevents the muscle to be fully activated, and thus, that central alterations are present. Because the muscle is never fully activated [START_REF] Herzog | Twitch interpolation represents muscle activation in a qualitative manner only[END_REF], according to the literature, a level of activation voluntary activation ≥ 90% in the fresh muscle can be considered as satisfactory [START_REF] Lepers | Neuromuscular fatigue during a long-duration cycling exercise[END_REF][START_REF] Millet | Mechanisms contributing to knee extensor strength loss after prolonged running exercise[END_REF][START_REF] Sidhu | Cortical voluntary activation of the human knee extensors can be reliably estimated using transcranial magnetic stimulation[END_REF]Giandolini et al., 2015;[START_REF] Mira | Cortical voluntary activation testing methodology impacts central fatigue[END_REF][START_REF] Twomey | La fatigue neuromusculaire au cours de l'exercice : considérations méthodologiques, étiologie et rôle potentiel dans la fatigue chronique[END_REF] and could be obtained also in old or very old adults [e.g. (Sundberg et al. 2018b)].,Voluntary activation levels lower than 80% can be expected in pathological populations or in injured athletes during the rehabilitation phase [START_REF] Shield | Assessing Voluntary Muscle Activation with the Twitch Interpolation Technique[END_REF]. Low levels of activation in healthy subjects would indicate the presence of fatigue, lack of motivation or errors in measurement (e.g. position of the subject or the use of compliant instrumentation). The accuracy can increase, for example, with the introduction of a familiarization session in the protocol. Error in the calculation of voluntary activation could be induced if the stimulation is not delivered on the plateau of MVIC. In that case, a correction in the original equation can be applied [START_REF] Strojnik | Neuromuscular fatigue after maximal stretch-shortening cycle exercise[END_REF]:

𝑣𝑜𝑙𝑢𝑛𝑡𝑎𝑟𝑦 𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 (%) = 100 -𝐷 * (𝑇𝑏/𝑇 𝑀𝐴𝑋 )/𝑃𝑂𝑇 * 100 Equation 2
Where Tb is the torque developed just before the stimulation, TMAX is the maximal torque obtained from the MVIC, POT is the amplitude of the potentiated twitch and D is the difference between Tb and the maximal torque obtained with the SIT.

Another potential limitation is the position of the muscle and the role of the series elastic slackness, which can be reduced by positioning the muscle at longer lengths or applying high-frequency doublets (100Hz) [START_REF] Shield | Assessing Voluntary Muscle Activation with the Twitch Interpolation Technique[END_REF]. The use of doublets is recommended because they are less influenced by postactivation potentiation than single stimulations and avoid the effect of antidromic collisions and spinal reflexes, activating more likely fibers which are in refractory period during the first stimuli [START_REF] Duchateau | Stimulation conditions can improve the validity of the interpolated twitch technique[END_REF]. This leads to a more accurate and less variable response in voluntary activation assessment (Rozand et al. 2020). However, in specific populations like older adults single twitches are usually preferred to doublets because of stimulation discomfort [START_REF] Shield | Assessing Voluntary Muscle Activation with the Twitch Interpolation Technique[END_REF].

In case of single twitches, it seems that longer stimuli durations (2 ms) are better correlated with doublet responses [START_REF] Zarkou | Comparison of techniques to determine human skeletal muscle voluntary activation[END_REF].

Doublets can be delivered to the peripheral nerve with magnetic stimulation to limit discomfort.

Although the evoked responses are similar than that with electrical stimulations in young adults (Bachasson et al. 2013), this technique seems less effective with great skinfold thickness (Tomazin et al. 2011).

Magnetic stimulation can also be applied over the motor cortex to quantify voluntary activation through TMS [START_REF] Todd | Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation[END_REF]. Because the supraspinal excitability is lower at rest and increases during contraction, it is inappropriate to normalise the TMS superimposed twitch to a twitch evoked at rest [START_REF] Todd | Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation[END_REF]. The relationship between the TMS SIT and the voluntary force has been demonstrated to be linear from 50% to 100% MVIC. Thus, [START_REF] Todd | Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation[END_REF] tried to estimate the resting twitch by extrapolation of the evoked responses during contractions at 50%, 75% and 100%

MVC using this linear relationship (estimated resting twitch; ERT), showing that the linearity is maintained in the fatigued elbow flexors and concluding that this procedure can illustrate the impairments at corticospinal level [START_REF] Todd | Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation[END_REF][START_REF] Todd | Reproducible measurement of voluntary activation of human elbow flexors with motor cortical stimulation[END_REF]. Several authors used TMS voluntary activation to provide insights to the etiology of performance fatigability in different tasks and conditions (e.g. [START_REF] Temesi | Central fatigue assessed by transcranial magnetic stimulation in ultratrail running[END_REF][START_REF] Temesi | Are Females More Resistant to Extreme Neuromuscular Fatigue?[END_REF][START_REF] Zghal | Improved tolerance of peripheral fatigue by the central nervous system after endurance training[END_REF][START_REF] Jubeau | Neuromuscular Fatigue during Prolonged Exercise in Hypoxia[END_REF]. However, there are several methodological limitations. For example, the three evoked responses used to interpolate the ERT should present a linear correlation with a pearson's r coefficient >0.90 (Hunter et al. 2006;[START_REF] Cadigan | Maximal voluntary activation of the elbow flexors is under predicted by transcranial magnetic stimulation compared to motor point stimulation prior to and following muscle fatigue[END_REF]). However, this cannot usually be checked online, increasing the risk of missing data. Furthermore, the number of stimulation used to calculate the ERT and the long duration of the assessment procedures could influence the calculation of voluntary activation [START_REF] Mira | Cortical voluntary activation testing methodology impacts central fatigue[END_REF][START_REF] Dekerle | Methodological issues with the assessment of voluntary activation using transcranial magnetic stimulation in the knee extensors[END_REF]. More recently, the validity of the calculation of voluntary activation using TMS in the knee extensors has been questioned [START_REF] Nuzzo | Voluntary activation of knee extensor muscles with transcranial magnetic stimulation[END_REF]). These authors argued that the optimization of the stimulation intensity is challenging as the motor evoked responses of the antagonists (hamstrings) is rarely measured (due to technical difficulties to effectively stimulate the sciatic nerve). They also argued that the ERT calculated in the knee extensors is systematically underestimated because of the consistent differences between ERT and POT evoked via electrical stimulation both at rest and in fatigued condition. Finally, they questioned the effectiveness of TMS to activate an appreciable number of motor units of the KE motoneuronal pool, as the SITTMS/SITPNS ratio rarely overcome the value of 0.80 and the MEP size overcome the 50% of the M-wave evoked via electrical stimulation.

For those methodological reasons, TMS could not be an appropriate technique to study age-related changes in voluntary activation of the knee extensors. To date, no study showed age-related changes in this parameters when using TMS, contrary to some evidences showing lower voluntary activation in older adults using PNS (Rozand et al. 2020).

TAKE HOME MESSAGE: NON-INVASIVE MEASUREMENT OF PERFROMANCE

FATIGABILITY

The assessment and interpretation of fatigability and changes in the neuromuscular system need a clear and accepted methodology. Stimulation techniques coupled with force and EMG measures allow to quantify impairments in contractile function and/or voluntary activation in a non-invasive manner. The interpolated twitch technique is the most diffused procedure for evaluating the etiology of performance fatigability, and consists in delivering stimulations during and after an isometric maximal contraction. Stimulations can be delivered over the motor cortex (magnetic), over the motor nerve (magnetic and electrical) or over the muscle belly (electrical). Finally, duration and intensity of the stimulation can be adapted to the population and aim of the investigation.

NEUROMUSCULAR CHANGES WITH AGING

Neuromuscular alterations with age

The neuromuscular system properties and functionality changes with advancing age. The progressive reduction in muscle mass (sarcopenia) with age induces a decrease in maximal force and power output (dynapenia) (Harridge and Lazarus 2017). From ~35 to ~60 years old, 10-20% of total muscle mass is lost (Mitchell et al. 2012). A further ~20% is lost in the next twenty years, when approaching the ninth decade of life [START_REF] Vandervoort | Aging of the human neuromuscular system[END_REF]. Sarcopenia and dynapenia are both associated with increased risk of falls, hospitalisation, loss of autonomy, frailty and mortality (Newman et al. 2006;[START_REF] Cruz-Jentoft | Sarcopenia: Revised European consensus on definition and diagnosis[END_REF].

Neuromuscular deterioration could be due to neurodegenerative disorders or cognitive impairments [e.g. (D'Angelo and Bresolin 2006)], however, in healthy older adults, the primary cause of these age-related alterations are due to a progressive loss of motor-units (MU) [START_REF] Power | Human neuromuscular structure and function in old age: A brief review[END_REF]Hepple and Rice 2016;Hunter et al. 2016;McNeil and Rice 2018).

Properties of the motor units

The MU is usually described as the "final common path" of the nervous system (McNeil and Rice 2018), and consists in a α-motoneuron located in the spinal cord and all the individual muscle fibers innervated by this motoneuron [START_REF] D'angelo | Cognitive impairment in neuromuscular disorders[END_REF][START_REF] Power | Human neuromuscular structure and function in old age: A brief review[END_REF]. All muscle fibers innervated by the same motoneuron are of the same fiber type, and are spread over a relatively large area within the muscle, called neuromuscular compartment [START_REF] Peters | Structure and function in vertebrate skeletal muscle[END_REF]). This area can cover 15% to 75% of the muscle volume, depending on the muscle considered (D'Angelo and Bresolin 2006).

Usually, type I muscle fibers compose smaller MU called slow-twitch MU, with lower threshold for full activation [tetanus at ~20 Hz [START_REF] Enoka | Neuromechanics of Human Movement[END_REF]]. They are the most "fatigue-resistant" of the muscle fibers. On the other side of the spectrum, fast-twitch MU are large units composed by type IIx fibers, even if it is not excluded that they could be composed by type IIa fibers, depending on the method of categorization [START_REF] Enoka | Neuromechanics of Human Movement[END_REF]. Fast-twitch MU have an high full activation threshold [up to 80-100 Hz for a tetanus [START_REF] Enoka | Neuromechanics of Human Movement[END_REF]] and are mainly adopted during short and intense muscle action (e.g. jumping, sprinting). In between, medium-to-large size motor units, known as fast fatigue-resistant motor units, are intermediate MU and composed by type IIa fibers. Characteristics of the different types of muscle fibers are presented in Table 1. Adapted from: [START_REF] Bruton | Muscle Plasticity[END_REF]).

Age-related changes in the motor units

The loss in MU number with age, and thus muscle mass, is associated with a variety of factors that include DNA damage, alterations in blood flow, systemic inflammation, reduction in neurotrophic signalling, and decreased Schwann cell function leading to impairments in myelinated fibers (αmotoneuron) (Allen et al. 2021). The MU loss starts with the denervation of the α-motoneuron from the muscle fibers (Allen et al. 2021), probably due to mechanisms such as deterioration and death by apoptosis of the α-motoneuron [START_REF] Faulkner | Age-related changes in the structure and function of skeletal muscles[END_REF], possible perturbations in the signaling network at the level of the neuromuscular junction or injury-related necrosis of the muscle fibers (Hepple and Rice 2016). With the loss of the α-motoneuron, the muscle fibers can either be lost or reinnervated and integrated in a proximal MU via motor axon sprouting [START_REF] Gordon | Adaptive and maladaptive motor axonal sprouting in aging and motoneuron disease[END_REF]). This axonal sprouting is a natural mechanism of the cycle of denervation-reinnervation of muscle fibers that occurs over the lifespan, for example to compensate for α-motoneuron losses following partial nerve injuries [START_REF] Siu | Mechanisms controlling axonal sprouting at the neuromuscular junction[END_REF].

The process of denervation-reinnervation accelerates with age, resulting into fewer but larger MU, increasing the number of fibers innervated from an average of 3 to 5-fold to a maximum of 8-fold [START_REF] Siu | Mechanisms controlling axonal sprouting at the neuromuscular junction[END_REF]. This imply that only a finite number of muscle fibers could be incorporated in by a MU, with the exceeding fibers being lost. This "saturation" phenomenon have been traced

back to the severe muscle atrophy of very old adults (i.e. > 80 years old), where muscle remodelling could be severely limited (McNeil and Rice 2018). Furthermore, larger MU have greater metabolic demands, which is related to increased oxidative stress and could result in an accelerated deterioration at advanced age [START_REF] Gordon | Adaptive and maladaptive motor axonal sprouting in aging and motoneuron disease[END_REF]McNeil and Rice 2018). Finally, despite the atrophy with age seems uniform between type I and II muscle fibers in humans [START_REF] Power | Human neuromuscular structure and function in old age: A brief review[END_REF], motoneuronal death seem to occur preferentially in large motor neurons (i.e. fast-twitch MU), at least in animal models [START_REF] Kanda | Changes in properties of the medial gastrocnemius motor units in aging rats[END_REF]. This would result, for example, in a reinnervation of type IIa With the age-related remodelling in MU, the neuromuscular system in old and very old adults would progressively perform with fewer and larger MUs, prevalently of slow-twitch type. This would result in higher reliance on the oxidative metabolism for the muscles of older adults. Fibers from the remaining MU in advanced age are typically smaller, with atrophy predominantly affecting large muscle mass such as the lower limbs [Figure 7, (Hunter et al. 2016)]. Deterioration in MU and loss in muscle mass are not uniform across individuals with age (Hunter et al. 2016). Indeed, heterogeneity in epigenetic factors, nutrition, hormonal status, inflammation status and disuse, increase betweensubjects variability (Hunter et al. 2016;Hunter 2018).

Changes in single muscle fibers function with age

The specific tension of the muscle fibers (force per unit) seems to be preserved with aging [START_REF] Venturelli | Skeletal Muscle Function in the Oldest-Old: The Role of Intrinsic and Extrinsic Factors[END_REF], although this is a debatable subject (Hunter et al. 2016). Conversely, contraction speed and relaxation rate seem to deteriorate, especially in very old adults [START_REF] Baudry | Postactivation potentiation influences differently the nonlinear summation of contractions in young and elderly adults[END_REF][START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF][START_REF] Power | Human neuromuscular structure and function in old age: A brief review[END_REF]Hunter et al. 2016). This is probably due to slower cross-bridge formation and rates of Ca 2+ uptake, and the reduced activity of the Ca 2+ -ATPase activity, although physical activity could reduce part of these age-related deteriorations [START_REF] Hunter | Human skeletal sarcoplasmic reticulum Ca2+ uptake and muscle function with aging and strength training[END_REF]. Finally, contractility of the muscle tissue could also be reduced by a progressive change in muscle architecture and fat infiltration within the contractile tissue (Figure 7), compromising muscle contractile properties of the whole muscle [START_REF] Klein | Normalized force, activation, and coactivation in the arm muscles of young and old men[END_REF][START_REF] Delmonico | Longitudinal study of muscle strength, quality, and adipose tissue infiltration[END_REF]).

Age-related alterations in motor drive and voluntary activation

Other than structural integrity, the performance of the neuromuscular system also depends on an adequate activation of the motor units [START_REF] Reid | Skeletal muscle power: A critical determinant of physical functioning in older adults[END_REF]Hunter et al. 2016;Hunter 2018).

The ability to rapidly activate and contract the muscle, assessed with the rate of force development, is impaired with advancing age [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF]Varesco et al. 2019a), suggesting alterations of the voluntary drive to the muscle during rapid contractions. The impaired ability to generate or to maintain high discharge frequencies could lead to different or less efficient activation pattern with aging [START_REF] Klass | Age-related decline in rate of torque development is accompanied by lower maximal motor unit discharge frequency during fast contractions[END_REF][START_REF] Maffiuletti | Rate of force development: physiological and methodological considerations[END_REF]Varesco et al. 2019a). Deteriorations of the motor nerve could also alter its excitability (Hunter et al. 2016) and the conduction velocity of the impulse [START_REF] Campbell | Physiological changes in ageing muscles[END_REF]. These alterations could also contribute to a decrease in the capacity to activate the muscles in older adults. However, previous studies showed moderate [e.g. [START_REF] Stevens | Are voluntary muscle activation deficits in older adults meaningful?[END_REF][START_REF] Škarabot | Reduced corticospinal responses in older compared to younger adults during submaximal isometric , shortening and lengthening contractions[END_REF]] or no impairments [e.g. [START_REF] Roos | Quadriceps muscle strength, contractile properties, and motor unit firing rates in young and old men[END_REF]] in voluntary activation with age. In a recent meta-analysis pooling all the studies comparing voluntary activation level in young and old adults, Rozand et al. ( 2020) evidenced a moderate reduction in voluntary activation in older adults.

Moreover, older adults are more variable across trials in their activation level, with studies showing that older adults needs more practice and multiple trials to achieve a similar level of activation compared to young adults [START_REF] Jakobi | Voluntary muscle activation varies with age and muscle group[END_REF][START_REF] Hunter | Recovery from supraspinal fatigue is slowed in old adults after fatiguing maximal isometric contractions[END_REF]. Changes in voluntary activation level depends on muscle groups and techniques adopted, with higher likelihood to observe age-related impairments with electrically evoked high-frequency doublets and trains of stimuli [START_REF] Klass | Voluntary activation during maximal contraction with advancing age: A brief review[END_REF]Rozand et al. 2020). The decline in motor drive to the motor units pool might reflect a neural rate [START_REF] Christie | Short-term training adaptations in maximal motor unit firing rates and afterhyperpolarization duration[END_REF][START_REF] Unhjem | Lifelong strength training mitigates the age-related decline in efferent drive[END_REF]) to a similar level than young adults [START_REF] Hansen | Effects of Resistance Training Frequency on Short-Term Neuromuscular Adaptations in Untrained Participants[END_REF]. Overall, impairments in voluntary activation seem modest compared to alterations in muscle mass or maximal force but may be underestimated in a laboratory setting, where multiple attempt and familiarization are a common practice [START_REF] Jakobi | Voluntary muscle activation varies with age and muscle group[END_REF]Rozand et al. 2020).

Muscle function: force and power

From the fourth to the ninth decade of life, humans can lost up to 40% of their maximal strength [START_REF] Lindle | Age and gender comparisons of muscle strength in 654 women and men aged 20 -93 yr[END_REF]. In very old age, force and power loss range approximatively from -1.5% to -3% every year [START_REF] Skelton | Strength, power and related functional ability of healthy people aged 65-89 years[END_REF]Mitchell et al., 2012). Because of the large between-subjects variability, longitudinal studies suggest that these values may be underestimated [START_REF] Frontera | Aging of skeletal muscle: a 12-yr longitudinal study[END_REF][START_REF] Hughes | Longitudinal muscle strength changes in older adults: Influence of muscle mass, physical activity, and health[END_REF]. The lower limbs muscle, in particular, are greatly affected by the age-related loss in muscle force and power [START_REF] Ditroilo | Effects of age and limb dominance on upper and lower limb muscle function in healthy males and females aged 40-80 years[END_REF]. For example, [START_REF] Hughes | Longitudinal muscle strength changes in older adults: Influence of muscle mass, physical activity, and health[END_REF] investigated the changes in maximal strength of the knee and elbow extensors in both men and women (46 to 78 years old) over ~10-years. These authors found a loss of ~14% of isokinetic strength per decade for knee extensors in both sexes. For the elbow extensors, men and women presented a decline of ~12% and ~2% per decade, respectively. Interestingly, these authors observed that the decrease in muscle strength was ~60% larger than the estimated loss from a cross-sectional study on the same population.

Contraction mode affects the age-related differences

The changes in maximal force with aging varies across muscles [START_REF] H829 Winegard | A 12-Year Follow-up Study of Ankle Muscle Function in Older Adults[END_REF][START_REF] Hughes | Longitudinal muscle strength changes in older adults: Influence of muscle mass, physical activity, and health[END_REF][START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF]) and tasks performed [START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF]Hunter et al. 2016). The difference between young and older adults is greater in concentric vs. isometric and eccentric contractions [START_REF] Klass | Aging does not affect voluntary activation of the ankle dorsiflexors during isometric, concentric, and eccentric contractions[END_REF][START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF]. For concentric contractions, the age-related impairments in maximal torque increases with contraction velocity in the knee extensors, but not in the ankle dorsiflexors [START_REF] Lanza | Effects of age on human muscle torque, velocity, and power in two muscle groups[END_REF][START_REF] Klass | Aging does not affect voluntary activation of the ankle dorsiflexors during isometric, concentric, and eccentric contractions[END_REF]. Because of the lower contractile speed due to muscle remodelling with advancing age, maximal contraction speed is slower in old than young adults, with further impairments in very old adults (Sundberg et al. 2018b). Older adults show ~15-40% lower maximal contraction velocities compared to young adults [START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF], with some healthy older adults not being able to develop fast contraction velocities [e.g. 270°.s -1 , (Callahan and Kent-Braun 2011)]. Because of the loss in maximal velocity and maximal force with age, there is a change in the force-velocity relationship that greatly affects maximal power production (Figure 8).

Indeed, the decline in maximal power surpasses the decline in maximal isometric or concentric strength [START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF]. When eccentric contractions are performed, maximal torque deficit has been shown to be less pronounced than during isometric and concentric contractions [e.g. [START_REF] Hortobágyi | The influence of aging on muscle strength and muscle fiber characteristics with special reference to eccentric strength[END_REF][START_REF] Lindle | Age and gender comparisons of muscle strength in 654 women and men aged 20 -93 yr[END_REF][START_REF] Klass | Aging does not affect voluntary activation of the ankle dorsiflexors during isometric, concentric, and eccentric contractions[END_REF][START_REF] Rozand | Voluntary activation and variability during maximal dynamic contractions with aging[END_REF]]. This difference seems independent of muscle mass, muscle fiber type or fiber size [START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF]. Despite the underlying mechanisms remain to be fully understood [START_REF] Raj | Aging and the force-velocity relationship of muscles[END_REF], it has been suggested that the age-related increase in intramuscular connective tissue could increase passive resistance during muscle lengthening [START_REF] Hortobágyi | The influence of aging on muscle strength and muscle fiber characteristics with special reference to eccentric strength[END_REF]. Another possible explanation could be that the slower crossbridge formation and thus slower rate of force development might provide some advantages during eccentric contractions [START_REF] Porter | Eccentric peak torque of the plantar and dorsiflexors is maintained in older women[END_REF]. Finally, studies in vitro also found that passive properties were preserved in the muscle fibers of older adults compared to young adults [START_REF] Ochala | Single skeletal muscle fiber behavior after a quick stretch in young and older men: A possible explanation of the relative preservation of eccentric force in old age[END_REF].

Sex-related differences in muscle function with age

In general, men possess larger skeletal muscle mass than women and possess, at least for some muscle groups, a larger percentage of type II muscle fibers, that makes men rely more on anaerobic energetic metabolism [START_REF] Hunter | Sex differences in human fatigability: Mechanisms and insight to physiological responses[END_REF]. The loss in maximal strength with age seems not affected by sex in the knee extensors (Haynes et al. 2020). However, alterations in maximal concentric force at 60°.s -1 accelerate earlier in women (~40 years old) than men (~70 years old) (Haynes et al. 2020). Men shows a steep decline at very old age that reduces the sex-related differences in concentric force production (Haynes et al. 2020). However, eccentric torque might be better preserved in older women than men, possibly be due to the greater capacity of women to store and utilize elastic energy per unit of muscle or to the lower absolute force generated by women [START_REF] Lindle | Age and gender comparisons of muscle strength in 654 women and men aged 20 -93 yr[END_REF][START_REF] Klass | Aging does not affect voluntary activation of the ankle dorsiflexors during isometric, concentric, and eccentric contractions[END_REF]. Specific age-related alterations in performance fatigability are discussed in chapter 4.

TAKE HOME MESSAGE: NEUROMUSCULAR CHANGES WITH AGING

Aging induces both structural and functional alterations in the neuromuscular system, including a remodeling and loss of motor units. However, impairments in the capacity to develop force and power are even greater, especially in very old adults. Despite alterations in the ability to rapidly develop force in older adults, which could be due to impairments in the neural drive to the muscle, maximal

AGE-RELATED ALTERATIONS IN PERFORMANCE FATIGABILITY

The age-related deterioration in muscle structure and activation capacity can have a major impact on activities of daily living that require repeated or prolonged exertion. The loss in muscle force and power with age would imply that simple activities like walking or gardening are performed at a higher percentage of maximal capacity in old compared to young adults, possibly influencing performance fatigability.

Performance fatigability is important to consider for quality of life in older adults. For example, the activities of daily living could progressively induce fatigue along the day, reducing the ability to perform motor tasks [e.g. [START_REF] Murphy | Ecological measurement of fatigue and fatigability in older adults with osteoarthritis[END_REF]]. Similarly to the loss of strength and power, agerelated differences in performance fatigability depend on the task characteristics, such as muscle groups involved, intensity, or mode of contraction (Hunter et al. 2016;Hunter 2018;Krüger et al. 2018).

Isometric tasks

Isometric contractions fatiguing tasks are commonly used to assess performance fatigability in various populations using sustained or intermittent contractions. Because maximal strength is lower in old compared to young adults, the contraction intensity is usually set relatively to maximal isometric force to normalize the workload. In this condition, healthy older adults are typically less fatigable than young adults for both maximal and submaximal isometric fatiguing tasks [e.g. (Callahan et al. 2009;Solianik et al. 2017)]. This could be explained by their higher percentage of slow-twitch motor units, or lower metabolite accumulation (e.g. inorganic phosphate, changes in pH) due to lower occlusion of the muscle mass (Moritani et al. 1992). The important role of muscle occlusion and metabolite accumulation is underlined by studies evaluating sex-related differences in the plantar flexors (Russ and Kent-Braun 2003), which found lower performance fatigability in freeflow condition for women than men, but similar performance fatigability when blood flow was occluded. It was argued that larger muscle mass in men compared to women produces higher intramuscular pressure, decreasing oxygen delivery and increasing metabolite accumulation because of muscle ischemia induced by the isometric contractions (Russ and Kent-Braun 2003). However, when repeating similar experiments comparing young and older adults, the age-related difference in performance fatigability was maintained even during ischemia [START_REF] Chung | Age-related resistance to skeletal muscle fatigue is preserved during ischemia[END_REF]).

The results reported by [START_REF] Chung | Age-related resistance to skeletal muscle fatigue is preserved during ischemia[END_REF] suggest that oxygen supply might not be the main responsible for the lower fatigability per unit of muscle observed in older adults. Another possible explanation could be the slowing in muscle contractile properties in older adults that might result in lower MU firing rates and, consequently, less energy expenditure during a sustained isometric task [START_REF] Connelly | Motor unit firing rates and contractile properties in tibialis anterior of young and old men[END_REF]Tevald et al. 2010). A model reporting the hypothetical causes of the increased metabolic economy in skeletal muscle in the older adult proposed by Kent-Braun ( 2009) is presented in Figure 9.

Importantly, lower performance fatigability for older adults is not consistently reported when using isometric protocols (Krüger et al. 2018). For example, when isometric intermittent protocols (5-s on/5-s off) were used, old adults and young adults showed similar performance fatigability on the knee extensors (Stackhouse et al. 2001b), while lower fatigability was observed for older adults in other muscle groups [e.g. (Kent-Braun et al. 2002;[START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF]].

Regarding very old adults, because of the great loss in MU and atrophy of muscle fibers, this relative fatigue resistance could be possibly lost or reduced. There is a limited body of evidence investigating this hypothesis, showing that very old adults were similarly (Allman and Rice 2001) or more (Justice et al. 2014) fatigable than young adults, depending on the muscle considered. Because the lower limbs are particularly affected by the age-related deterioration in muscle function [START_REF] Janssen | Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr[END_REF][START_REF] Hughes | Longitudinal muscle strength changes in older adults: Influence of muscle mass, physical activity, and health[END_REF][START_REF] Ditroilo | Effects of age and limb dominance on upper and lower limb muscle function in healthy males and females aged 40-80 years[END_REF], the knee extensors performance fatigability in old and very old adults is of great interest. The function of the knee extensors is essential for everyday life activities such as walking [START_REF] Hurley | Quadriceps function, proprioceptive acuity and functional performance in healthy young, middle-aged and elderly subjects[END_REF]) and has been linked to the risk of fall and frailty (e.g. [START_REF] De Rekeneire | Is a fall just a fall: Correlates of falling in healthy older persons. The health, aging and body composition study[END_REF][START_REF] Pijnappels | Identification of elderly fallers by muscle strength measures[END_REF]). Yet no evidence exists on the knee extensors performance fatigability of very old adults after isometric fatiguing tasks. In both young and older adults, performance fatigability observed after isometric fatiguing tasks seem mainly explained by impairments in contractile function, with a relatively small decrease in voluntary activation (Krüger et al. 2018). After a 2-min sustained isometric contraction of the knee extensors, Solianik et al. (2017) observed greater impairments in contractile function and voluntary activation in young adults compared to old adults (Solianik et al. 2017), which could be explained by the large accumulation of metabolites and blood flow occlusion derived from the sustained high-intensity contraction. After an isometric intermittent task of the knee extensors for 4 minutes (5-s on/ 5-s off, maximal contractions), Callahan et al. (2009) observed greater impairments in contractile function in young than old adults, with no age-related difference in voluntary activation. The total work performed was greater in young than old adults, and the origin of performance fatigability was more attributed to impairments in contractile function than voluntary activation (Callahan et al. 2009).

Taken together, those results suggest that, after an isometric fatiguing task of the knee extensors performed at an intensity relative to the maximal capacity, older adults show lower performance fatigability compared to young adults (Callahan et al. 2009;Solianik et al. 2017). The magnitude of this difference seems more mediated by alterations in contractile function than impairments in voluntary activation (Callahan et al. 2009;Solianik et al. 2017). While relative workloads could provide useful insights on the properties of the neuromuscular system, it could lead to some misinterpretation of the results, with limited transferability to activities of daily living (Hureau et al. 2021).

Studies exploring performance fatigability of the knee extensors in isometric condition using absolute workloads and including the investigation of very old adults have yet to be performed.

Concentric tasks

Concentric mode of contraction requires an active shortening of the muscle, which is closer to what is required from the activities of daily living (Figure 10). The majority of the studies investigating neuromuscular function of the knee extensors evaluated single limb tasks in both isotonic and isokinetic tasks, setting the workload as a percentage of maximal capacity. These studies showed that older adults are typically more fatigable than young at high contractions speed (e.g. >180°.s -1 ) during both isokinetic maximal contractions at 270°.s -1 (Callahan and Kent-Braun 2011) and isotonic maximal velocity contractions at 20% of maximal isometric force (Dalton et al. 2012;Senefeld et al. 2017;Sundberg et al. 2018bSundberg et al. , 2019)). However, at slower contraction speed (80-180°.s -1 ), similar performance fatigability was observed (Callahan and Kent-Braun 2011;Dalton et al. 2012). At 80°.s - 1 ), performance fatigability was higher for the young adults (Weavil et al. 2018). When young and old adults performed a similar workload for a similar duration (iso-work condition), however, performance fatigability was lower in young compared to older adults (Weavil et al. 2018). The modality of the concentric contraction (isokinetic vs. isotonic) could also influence performance fatigability. Dalton et al. (2015) compared the age-related differences following an isokinetic (fixed velocity) vs. isotonic (fixed load) fatiguing task performed until 40% of the maximal power was lost (Dalton et al. 2015). Older adults performed 20% fewer contractions at the isotonic task compared to the young, suggesting that the lower velocity production capacity might not explain the difference in performance and fatigability with age during concentric tasks. Indeed, no age-related differences were found for the isokinetic task (Dalton et al. 2015).

Regarding very old adults, after a fatiguing exercise at maximal speed during isotonic contractions (20% MVIC), very old adults showed greater impairments in force and power production than both old and young adults independent of sex (Sundberg et al. 2018b), suggesting accentuated impairments with advancing age. Whether or not the difference in performance fatigability between old and very old adults varies across different tasks remains unknown.

Studies evaluating the etiology of performance fatigability with concentric contractions suggest peripheral rather than central impairments with age (Dalton et al. 2015;Sundberg et al. 2018a, b;Weavil et al. 2018). Evidence of a reduced contraction efficiency have been shown in concentric mode with aging, especially in very old adults (Layec et al. 2018). This age-related impairment in contraction efficiency in concentric tasks does not seem to be associated with altered oxygen supply, as similar levels of tissue oxygenation (Layec et al. 2018) and blood flow (Weavil et al. 2018) were found in young and older adults. This would also suggest that differences in contraction economy between isometric and concentric contractions would not be due to oxygen supply. By studying the age-related differences in muscle bioenergetics of the knee extensors, Sundberg et al. (2019) did not observe any age-related differences in oxidative capacity during the concentric task, but greater metabolite accumulation (Pi, H +, and dihydrogen phosphate) within the working muscle in older adults compared to young adults. These results suggest that the lower contractile economy would lead to higher metabolite accumulation, inhibiting directly the contractile function within the muscle. The fatigability observed in the knee extensors after concentric fatiguing tasks are associated with both walking endurance and balance in the elderly [START_REF] Reid | Skeletal muscle power: A critical determinant of physical functioning in older adults[END_REF]. However, further insights can be obtained by evaluating multiarticular or locomotor fatiguing tasks. When normalizing for the individual one-repetition maximum at the leg press, old and young adults showed similar performance fatigability, but lower total work performed for older adults [START_REF] Hakkinen | Neuromuscular fatigue and recovery in women at different ages during heavy resistance loading[END_REF][START_REF] Bieuzen | Age-related changes in neuromuscular function and performance following a high-intensity intermittent task in endurance-trained men[END_REF]. During a cycling task at 80% of maximal power, Weavil et al. (2018) reported greater performance fatigability in young than older adults, which was accompanied by greater impairments in voluntary activation and contractile functions for young adults. However, when absolute intensity and duration were matched between age groups, young adults presented lower performance fatigability and lower impairments in contractile function and voluntary activation compared to old adults, indicating that absolute performance and fatigability are impaired with age during a cycling task (Weavil et al. 2018). Krüger et al. (2020) investigated performance fatigability and its etiology following cycling fatiguing tasks of various intensities. They found similar levels of performance fatigability after a sprint (Wingate) and a long moderate-intensity task, while performance fatigability was greater for young than older adults when exercising at severe intensity (Krüger et al. 2020). Because young adults produced more total work than old adults for all fatiguing tasks, these authors suggested a relationship between performance at the cycling tasks, power development capacity, and performance fatigability (Krüger et al. 2020). Other than power development capacity, performance in older adults could be limited by the reduced maximal aerobic capacity and oxygen delivery caused by the decrease in cardiac output and maximal heart rate responses during exercise (Wilson and Tanaka 2000). This would lead to a higher utilization of anaerobic metabolisms and, because anaerobic capacities decrease with aging [e.g. [START_REF] Reaburn | Anaerobic performance in masters athletes[END_REF]], exercise cessation. Because the cardiovascular system is more stressed during a multiarticular (cycling) than a single-limb (isokinetic)

task [e.g. (Weavil et al. 2018)], the reduction in cycling performance in old compared to young adults do not depend solely on impairments of the neuromuscular system (Poole et al. 2016;Krüger et al. 2020). Whether or not differences in performance and fatigability between a single-limb vs.

multiarticular tasks exist in very old compared to older adults remains to be investigated.

TAKE HOME MESSAGE: AGE-RELATED ALTERATIONS IN FATIGABILITY

The age-related differences in performance fatigability vary across tasks. Older adults show lower performance fatigability after isometric fatiguing tasks. Those differences are attenuated during intermittent tasks that reduce the negative effects of blood flow occlusion. During isokinetic and isotonic contractions, performance and performance fatigability are impaired with age, with greater impairments at faster velocities. Finally, when performing multiarticular tasks like cycling, performance and performance fatigability impairments with age depend on the duration and intensity of exertion, with the cardiovascular system contributing greatly to these age-related differences during intense and prolonged efforts. The benefits of physical activity are also evident at the MU level, with active individuals presenting more stable neuromuscular transmission (Power et al. 2016a) and greater slow twitch muscle fibers power [START_REF] Gries | Single-muscle fiber contractile properties in lifelong aerobic exercising women[END_REF]. MU number preservation in active older adults is still controversial, with greater preservation observed in the tibialis anterior by [START_REF] Power | Motor unit number estimates in masters runners: Use it or lose it?[END_REF], although not by others [START_REF] Piasecki | Motor unit number estimates and neuromuscular transmission in the tibialis anterior of master athletes: evidence that athletic older people are not spared from age-related motor unit remodeling[END_REF]. There is also minimal preservation of the number of estimated MU in the vastus lateralis [START_REF] Piasecki | Long-term endurance and power training may facilitate motor unit size expansion to compensate for declining motor unit numbers in older age[END_REF]) and the biceps brachii [START_REF] Power | Motor unit survival in lifelong runners is muscle dependent[END_REF]) muscles.

As for young adults, the type of physical activity triggers specific adaptations in older individuals.

For instance, strength does not seem to be preserved in endurance-trained older adults [START_REF] Chambers | Skeletal muscle size, function, and adiposity with lifelong aerobic exercise[END_REF]. Conversely, strength-trained old adults typically show preserved muscle strength and power (less of an age-related decline) when compared with inactive old adults [START_REF] Mckendry | Muscle morphology and performance in master athletes: A systematic review and meta-analyses[END_REF]). This relative preservation of strength and power is associated with greater limb muscle mass and neural drive to the contracting muscles than inactive older adults [START_REF] Unhjem | Lifelong strength training mitigates the age-related decline in efferent drive[END_REF], counteracting sarcopenia and the reduced neural drive that could occur with advanced aging [START_REF] Christie | Short-term training adaptations in maximal motor unit firing rates and afterhyperpolarization duration[END_REF]Rozand et al. 2020). Taken together, these results indicate that the type of physical activity (endurance vs. resistance exercises) and quantity (active vs. non-active adults) need to be carefully evaluated and quantified when studying age-related changes in physiological function. Despite those differences between active and inactive older adults, physical activity is rarely used to discriminate participants such as sex or age, potentially masking the age-related differences between young and old adults in their physiological outcomes and function. (Varesco et al. 2021b). Future studies should quantify physical activity to separate or include only active or inactive older adults in order to differentiate the effects of inherent aging from those of physical inactivity.

OBJECTIVES AND HYPOTHESES

Aging is associated with a progressive deterioration of the neuromuscular system that could result in impairments in performance fatigability. These age-related alterations seem exacerbated at very old age and could lead to impaired mobility, loss of autonomy and frailty. Moreover, it is important to study the function of large muscle masses used in locomotion such as the knee extensors, which are particularly affected by aging and are related to physical function. Furthermore, the effect of physical activity level could mediate some of these age-related alterations, with evidence showing differences in performance between active and inactive older adults. Including only active older adults would minimize the negative effects of physical inactivity.

The general aim of the thesis was to investigate performance fatigability of the knee extensors in young, old and very old active adults. Specifically, the present work aimed to:

-Characterize the neuromuscular, functional (using standard clinical tests) and biological characteristics (using muscle biopsies) of the knee extensors of healthy very old men and women and evaluate their association with habitual physical activity level (study 1)

-Evaluate performance fatigability in healthy young, old and very old men and women using a standardized isometric fatiguing protocol (study 2)

-Extend those results to more ecologically valid conditions by comparing performance fatigability after isometric, concentric and cycling tasks with a workload set relatively to individual body weight (study 3).

Study 1 aimed to evaluate whether habitual physical activity is associated with the knee extensors performance (i.e. isometric strength and fatigability) and biological parameters (muscle structural, microvascular and metabolic properties) in healthy very old men and women. Fatigability was evaluated with an intermittent isometric incremental test. Biological parameters were assessed using muscle biopsy in the vastus lateralis muscle. Finally, the study explored the sex-related differences in the analyzed parameters. We hypothesized that a greater physical activity level would be associated with higher capillarization, enzymatic activity and maintenance of type IIA and IIX fibers in both very old men and women, and lower knee extensors fatigability. We did not expect however, an association between physical activity and knee extensors maximal force, according to previous evidence on older adults [START_REF] Chambers | Skeletal muscle size, function, and adiposity with lifelong aerobic exercise[END_REF]..

Stimulations

Magnetic and electrical stimulations were applied to the femoral nerve, which innervate the knee extensor muscles.

Femoral nerve magnetic stimulation

Femoral nerve magnetic stimulation (FNMS) was performed for study 1 and 2. FNMS was delivered through a 45-mm figure-of-eight coil powered by two linked Magstim 200 stimulators (peak magnetic field: 2.5 T, stimulation duration: 0.1 ms; Magstim, Whitland, UK). The coil was positioned above the femoral triangle to recruit the femoral nerve and all stimuli were given at 100% of the maximum stimulator output. Optimal stimulation site evoking the highest twitch force amplitude was determined with minor adjustments and marked on the skin. FNMS supramaximality was checked with decreasing stimulator power output (100%, 95%, and 90). Because the capacity of FNMS to deliver supramaximal stimuli is altered when fat thickness below the coil increases (Tomazin et al. 2011), clear plateaus in maximal twitch could not be observed for all participants. Thus, only valid responses to FNMS were retained for analysis. Stimulation intensity at the plateau was then increased by 30% to ensure supramaximality.

Physical activity

Participants wore an accelerometer on their wrist (wGT3X-BT, ActiGraph, Pensacola, USA) for at least 7 complete days (> 8 h of wear time per day), including one weekend [START_REF] Migueles | Accelerometer Data Collection and Processing Criteria to Assess Physical Activity and Other Outcomes: A Systematic Review and Practical Considerations[END_REF]Varesco et al. 2021b). Additionally, for study 1 daily energy expenditure was calculated using the Questionnaire d'Activité Physique de Saint-Etienne, a validated self-administered physical activity questionnaire that represented retrospectively the activity done over a period of one week [START_REF] Bonnefoy | Validation of a physical activity questionnaire in the elderly[END_REF][START_REF] Garet | A questionnaire-based assessment of daily physical activity in heart failure[END_REF]).

FATIGUING AND FUNCTIONAL TASKS

2.1 Fatiguing tasks

The isometric quadriceps intermittent fatigue test

The isometric quadriceps intermittent fatigue test (QIF test, study 1 and 2) consisted of incremental sets of 10 intermittent (5-s on / 5-s off) isometric contractions, starting at 10% MVIC for the first set with increments of 10% MVIC per set until exhaustion, as described by Bachasson et al. (Bachasson et al. 2013) (Figure 17). Visual feedback of the target force level was provided (Figure 12), and participants listened to a soundtrack with the contraction-relaxation rhythm. The target force level was defined with a line of 4-N thickness (2 N under and 2 N above the target force). Exhaustion was defined as two consecutive contractions below the target force level for 2.5 s. Evaluations of maximal force and responses to stimulation were performed before the test, immediately (~2 s) after the end of each set and at task failure. The duration between the end of a set and the beginning of the following one was set at 25 s. Starting from the second stage, the increments were set at 5% BW. The target load was displayed on a large screen using a 2-Nm thickness green line (1Nm above -1Nm below). Participants were instructed to follow the rhythm of contraction imposed by a metronome and to reach the line at each contraction, without surpassing the force level required. Stages were separated by 1 min. At the end of each stage and at exhaustion, an evaluation of maximal torque and responses to stimulation was performed (~10-s) and the rate of perceived exertion was asked on a 6-20 Borg scale (Borg 1982).

Task failure was defined as 5 consecutive contractions below the target line. Before and after the test, a series of 3 maximal voluntary concentric contractions was performed.

The concentric body-weight based QIF test

The body-weight based concentric QIF test (study 3) is a modified version of the concentric QIF test (Varesco et al. 2021d). In the original version, 100 contractions were performed (~ 160 s). We set the duration of the stages at 120 s to increase the number of neuromuscular evaluations performed due to large expected difference in performance between young and older adults based on pilot studies.

The load and protocol were identical to the isometric body-weight based QIF test [stages of 75 contractions (0.8 s on/0.8 s off; stage duration = 2 min) starting at 10% of body weight, with increments of 5%]. The range of motion for the concentric contractions was from 90° to 150°, with 180° corresponding to full-extension of the knee, and the velocity set at 120°.s -1 . The return to the starting position was passive and performed at the same speed. Participants were instructed to start the contraction immediately after the machine returned the leg to the starting position.

The duration of the duty cycle (0.8 s on/0.8 s off) was measured during pilot studies and was due to the intrinsic delay of the device to attain 120°.s -1 . The duration was consistent throughout the exercise and the 75-contractions stages lasted systematically 120 s.

The body-weight based QIF test on the customized cyclo-ergometer

The body-weight based QIF test on the cyclo-ergometer (study 3) consisted of 120-s stages of continuous cycling (37.5 rpm) starting at a load that corresponded to 10% of body weight. Due to different configuration of the experimental setting and consequent different lever arm, it was not possible to calculate the load similarly to the other two tests. The initial load was set at 0.6 W.kg -1 and successive increments at 0.3 W.kg -1 , based on pilot studies. The cadence was set at 37.5 rpm (75 pedal stroke for the 120-s duration of each stage) to match duty cycle for the three body-weight based QIF tests. Participants were provided visual feedback of the cadence and a metronome dictated the rhythm. Immediately after each 120-s stage, the pedals were blocked and participants performed a neuromuscular evaluation, and rate of perceived exertion was recorded. Task failure was defined as a cadence below 37.5 rpm for 8 s.

Functional capacity assessment

Functional capacity assessment included the Time-up-and-Go test (TUG, study 1 and study 3) and the 6-minute walk test (6MWT). For the TUG test, participants were required to stand up from a chair, walk a distance of three meters, turn around, return, and sit down on the chair (Mathias et al. 1986). The stopwatch was started when the back of the participants moved from the chair and stopped when the participants were seated on with their back on the back of the chair. For the 6MWT participants were instructed to cover the longest distance possible in 6 min on a corridor of 30 m (selfpaced), and feedback on the time elapsed was provided every minute (Crapo et al. 2002).

ADDITIONAL

SUPPLEMENTAL MATERIAL S1

Detailed procedures for histological analysis (Fiber type, cross section area and shape),

capillary identification and analysis and enzymatic activity analysis

Fiber type, cross-section area (CSA) and shape.

Two serial cross-sections were labeled with monoclonal antibodies against myosin heavy-chain-I [perimeter 2 /(4π*CSA)] was calculated, with a value of 1.0 indicating a circle and a value > 1.0 an increasingly elongated ellipse (Kirkeby and Garbarsch 2000;Bruusgaard et al. 2006;Verdijk et al. 2007;Gueugneau et al. 2015). Finally, the area occupied by each fiber type for 100 fibers was obtained by multiplying the fiber type distribution to the fiber-type specific cross-sectional area (%A).

For all participants, fiber type proportion and morphometry were assessed for an average of 300 fibers per individual.

Capillaries

The identification of capillaries was performed using the monoclonal anti-CD31 (M0823, Dako, Glostrup, Danemark), which recognizes platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane glycoprotein strongly expressed by vascular endothelial cells (Charifi et al. 2004).

Cross-sections were labelled with anti-CD31, co-labelled with anti-laminin-α1, and resolved with corresponding secondary antibodies conjugated to Alexa-Fluor 488 or 546. CD31 and MHC images were matched and analyzed with ImageJ to analyze the capillary network as previously described (Gueugneau et al. 2016). A mean of 450 fibers and 800 capillaries were analyzed per subject.

Capillary density (CD) was expressed as the number of capillaries counted per square mm. Capillaryto-fiber ratio (C/F) was calculated as the ratio between the number of capillaries and the number of fibers present in the same area. In addition to global indices (CD, C/F), individual fiber indices were also determined. For each fiber type, the mean number of capillaries around a single fiber (CAF) was calculated. The capillary-to-fiber interface was estimated using LC/PF index, which is the length of contact (LC) between capillaries and the fibers divided by perimeter of the same fiber (PF).

Enzymes activity

Approximately 20 mg of muscle were homogenized in 400 µL of buffer (Manitol 225 mM, tris HCL 10 mM, saccharose 75 mM, EDTA 0•1 mM) with a tissue homogenizer (Ultra-Turrax T8 IKA-Werke, Staufen, Germany). Samples were centrifuged (20 min, 650 g, 4°C) and the supernatant was carefully collected. The pellet was re-homogenized in 2/3 of the previous buffer and centrifuged a second time (20 min, 650 g, 4°C) with the previously-collected supernatant. The protein concentration was determined with bovine serum albumin (BSA) as a standard (Pierce BCA Protein Assay Kit, Thermo Scientific, Waltham, MA) and the supernatants were [JT1] stored at -80°C until subsequent analyses were performed. Myokinase, creatine kinase, enolase, lactate dehydrogenase, citrate synthase, βhydroxyacyl-CoA dehydrogenase and second and fourth respiratory chain complex maximal activities (UI/mg of protein) were determined by absorbance with a multi-label microplate reader

SUPPLEMENTAL MATERIAL S2

Sex-related differences in blood parameters.

Blood samples Men (n=15) Women (n=15) P-value

Fibrinogen (g/L)

Summary of the study 1

The results of the present study showed that, in very old adults, physical activity is positively associated with the total work (absolute performance) performed at the QIF test for women, and greater maintenance of type IIX muscle fibers and performance at the Get-up-and-go test for men.

Because the level of physical activity was relatively homogeneous across our sample, our results indicated that small variation in habitual physical activity level, quantified as steps.day -1 , could have an influence on important parameters such as knee extensors performance (women) and performance at a functional test usually adopted in clinical settings (Get-up-and-go test; men). This underlines the need to take into consideration physical activity in aging studies (Varesco et al. 2021b). Because of the homogeneity in physical activity level and because healthy and active very old individuals do not represent the population of octogenarians, the effects of physical activity found in the present study might be underestimated.

In study 1 we also explored the possible association between neuromuscular and biological parameters. Interestingly, in women, while maximal knee extensors isometric strength was negatively associated with fat mass, it was positively associated with muscle oxidative properties. No such association were observed for men, despite they possess greater maximal force and showed higher performance. This could result in different age-related differences in men and women following prolonged efforts (i.e. following a fatiguing task). Furthermore, no biological parameter was associated with performance and performance fatigability in very old adults. To further understand the mechanisms of performance and performance fatigability with aging, non-invasive techniques such as stimulations techniques could provide useful information into the etiology of performance fatigability.

In the second study, we aimed to investigate the age-related differences in performance and performance fatigability in healthy and active men and women using the same QIF test paired with stimulation techniques.

Introduction

Healthy ageing is usually accompanied by impairments in neuromuscular function and fatigability, leading to increased risk of autonomy loss and incidence of frailty (Hunter 2018). These impairments are not linear with age. For example, the decline of maximal isometric force accelerates in very old adults (i.e. >80 years) compared to old adults (i.e. >60 years) (Mitchell et al. 2012;[START_REF] Venturelli | Skeletal Muscle Function in the Oldest-Old: The Role of Intrinsic and Extrinsic Factors[END_REF], with greater impairments in women compared to men (Haynes et al. 2020;Varesco et al. 2021a). In a recent meta-analysis, Krüger et al. (Krüger et al. 2018) reported an overall lower performance fatigability (exercise-induced maximal force loss) in older compared to young adults after isometric fatiguing tasks. However, these results are equivocal, with some studies reporting a large effect size (Hedges's g >2) and others reporting no age-related difference or greater fatigability in older adults (Krüger et al. 2018). Importantly, studies investigating performance fatigability after isometric tasks in very old adults are limited. Allman and Rice (Allman and Rice 2001) compared performance fatigability after an intermittent isometric fatiguing task of the elbow flexors between young and very old adults, and reported similar values between the two age groups. However, the absence of a group of older adults did not allow the rate of decline in neuromuscular function between old and very old adults to be determined. Justice et al. (Justice et al. 2014) showed that time to exhaustion during an isometric sustained fatiguing task of the ankle dorsiflexors was longer for older adults (65-75 yr) compared to young adults (<35 yr), with similar performance between young and very old adults (75-90 yr). However, performance fatigability was not investigated in this study, nor the mechanisms explaining these age-related differences, i.e. above (voluntary activation) or below (contractile function) the neuromuscular junction.

In old adults, impairments in voluntary activation following single-limb exercises seem to be similar compared to young adults (Krüger et al. 2018). Conversely, impairments in contractile function with fatigue show inconsistent results, with similar or lower alterations in old compared to young adults after isometric fatiguing tasks (Krüger et al. 2018). Again, evidence for alterations in voluntary activation and contractile function in very old adults are scarce. Sundberg et al. (Sundberg et al. 2018b) observed greater impairments in contractile function but not voluntary activation compared to young and old adults. Because variability increases with advancing age, standardized and validated testing procedures as well as assessments of physical activity levels are needed when comparing groups of different ages (Varesco et al. 2021b).

Thus, the present study aimed to investigate performance fatigability of the knee extensor (KE) muscles induced by the isometric quadriceps intermittent fatiguing (QIF) test in young, old and very old men and women. We expected similar performance fatigability in young and very old adults after the isometric intermittent fatiguing test, while older adults would show a lower performance fatigability. We further hypothesized lower impairments in contractile function in old adults than very old and young adults, without age-related differences in voluntary activation impairments.

Methods

Participants

Thirty young adults [15 men (YM); 15 women (YW)], 19 old adults [10 men (OM); 9 women (OW)] and 30 very old adults [15 men (VOM); 15 women (VOW)] participated in the study and were clearly informed on the experimental procedures prior giving their written consent. Participants were free from psychological, musculoskeletal and neurological disorders or disability, and were asked to avoid exercise and alcohol consumption for 24 h before each visit. All the procedures were approved by the Comité de Protection des Personnes and were performed in accordance with the declaration of Helsinki (2013; ClinicalTrials.gov identifier: NCT02675192). The University Hospital of Saint-Etienne (France) was the sponsor of this study.

Experimental protocol

Participants visited the laboratory on two separate occasions. During the first visit, all participants were familiarized with the neuromuscular testing procedures, and an accelerometer (wGT3X-BT;

ActiGraph, Pensacola, USA) was given to wear for 7 consecutive days to objectively assess physical activity (Varesco et al. 2021b). The old and very old adults also performed a 6-minutes-walk-test (6MWT), which consisted in covering the longest distance possible in 6 minutes in a corridor of 30 meters (Crapo et al. 2002). One week later during the second visit, KE function was evaluated using the isometric QIF test (Bachasson et al. 2013).

Experimental setup

Participants were seated upright on a custom-build chair with the hip angle set at 120° (180°= full extension) to facilitate coil placement over the femoral triangle (see below). Knee angle was fixed at 90°. The right leg was systematically evaluated other than for specific reasons (i.e. operated knee, unilateral pain). In these instances, the left leg was tested (1 OW, 2 VOM, 1 VOW). Extraneous movements of the upper body were minimized using belts across the waist and the thorax. Force of the KE was assessed using a force transducer (Omega Engineering Inc., Stamford, CT, USA) firmly attached over the malleoli, and digitized at a sampling rate of 2 kHz by PowerLab System (16/30-ML880/P, AD Instruments, Bella Vista, Australia).

Femoral nerve magnetic stimulation

Femoral nerve magnetic stimulation (FNMS) was performed with a 45-mm figure-of-eight coil powered by two linked Magstim 200 stimulators (peak magnetic field: 2.5 T, stimulation duration: 0.1 ms; Magstim, Whitland, UK). The coil was positioned above the femoral triangle to recruit the femoral nerve and all stimuli were given at 100% of the maximum stimulator output. Optimal stimulation site evoking the highest twitch force amplitude was determined with minor adjustments and marked on the skin. FNMS supramaximality was checked with decreasing stimulator power output (100%, 95%, and 90%). Because FNMS effectiveness in deliver supramaximal stimuli is altered when fat thickness under the coil increases (Tomazin et al. 2011), clear plateaus in maximal twitch amplitude were not observed for all participants. Thus, only valid responses to FNMS were retained for analysis (YM: 15; OM: 8; VOM: 10; YW: 15; OW: 9; VOW: 8).

Isometric QIF test

The session started with a warm-up consisting of three ~3-s contractions at 30%, 50% and 70% of the perceived maximal force. Successively, two 5-s maximal voluntary isometric contractions (MVICs) were performed, separated by 1-min of rest. A third MVIC was performed if the difference between the peak force values was >5%. Participants were instructed to push "as hard as possible" and strong verbal encouragement was provided. Visual feedback of the force signal was displayed on a screen positioned in front of the participants. Then, a neuromuscular evaluation (NME) was performed. NME consisted of a 5-s MVIC during which a 100-Hz doublet (Db100,s) was superimposed through FNMS, and a resting potentiated 100-Hz doublet (Db100) delivered ~2 s after relaxation. Once the baseline measures were performed, the QIF test started after at least 1 min of recovery.

The QIF test consisted of incremental sets of 10 intermittent (5-s on / 5-s off) isometric contractions, starting at 10% MVIC for the first set, with increments of 10% MVIC per set until exhaustion (Bachasson et al. 2013). Visual feedback of the target force level was provided, and participants followed a soundtrack with the contraction-relaxation rhythm. A line of 4-N thickness indicated the target force (2 N below and 2 N above). Exhaustion was defined as two consecutive contractions where force decreased below the target force level for 2.5 s. NME was performed immediately (~2 s) at the end of each set and at task failure. The duration between the end of a set and the beginning of the following one was standardized at 25 s.

Data analysis

Physical activity data were analyzed using ActiLife software (v6.13.4, ActiGraph, Pensacola, USA)

and quantified as steps•day -1 . All the remaining data were analyzed using Labchart software (v8, ADInstruments). MVIC was calculated as the average of the highest 0.5-s window of the contraction.

Performance fatigability was defined as the percentage of MVIC loss from baseline. Total work was calculated as the sum of the force-time integral of each contraction and indicated absolute performance at the QIF test. Total number of contractions performed indicated relative performance.

Voluntary activation (VA) was calculated from the formula: VA% = Db100,s/Db100 × 100. Impairments in contractile function were quantified as the change in Db100 from baseline.

Statistical Analysis

All variables are reported as mean ± SD in the text, tables and figures. . For all analyses, when significant effects were observed, Bonferroni correction was applied to post-hoc analyses. Significance was set at P ≤ 0.05. Statistical analysis was conducted using SPSS software (Version 23.0, IBM, Chicago, IL).

Results

Baseline measurements

Table 1 summarizes the participants' characteristics. Young and old adults walked significantly more steps•day -1 than very old adults (age effect, P < 0.001), with women being more active than men (sex effect, P = 0.013). Very old adults performed shorter distance at the 6MWT than old adults (age effect, P < 0.001) with women performing shorter distance than men (sex effect, P < 0.001). P < 0.001; Table 2). When normalized to body weight, MVIC showed no sex × age interaction (P = 0.545), but decreased with age (age effect, P < 0.001), being greater in men than women (sex effect, P = 0.006; Table 2). Absolute Db100 values at baseline showed significant sex × age interaction (P = 0.001). For men, Db100 significantly decreased with age (all P < 0.001; Table 2). For women, significant differences were found between YW and VOW, and between OW and VOW (all P < 0.001). No difference was observed between YW and OW (P = 0.407; Table 2). Db100 was different between sexes only in the young group (P < 0.001; Table 2). VA did not show main effects of age or sex (P = 0.278 and P = 0.098, respectively), nor age × sex interaction (P = 0.567; Table 2).

Performance fatigability

Among all the participants, 2 VOM and 4 VOW did not perform the QIF test due to knee pain. Thus, performance data were analyzed for 13 VOM and 11 VOW. Number of contractions was similar across sexes (P = 0.733) but differed with age (P = 0.008). Post-hoc analysis indicated that older adults performed more contractions than young (P = 0.046) and very old adults (P = 0.007), without differences between young and very old adults (P = 1.000, Figure 1A). No significant age × sex interaction was found for number of contractions (P = 0.218). Total work showed significant main effects of age (P < 0.001) and sex (P < 0.001), but no age × sex interaction (P = 0.949). Post-hoc analyses showed greater total work for young and old adults compared to very old adults, without differences between young and old adults, and greater total work for men compared to women (Figure 1B). No main effect of sex nor sex × stage, sex × age or sex × stage × age interactions were found for the changes in MVIC, Db100 and VA during and after the QIF test (all P > 0.05). Thus, performance fatigability data for men and women were pooled together.

A significant stage × age interaction was observed for the decrease in MVIC relative to baseline (P < 0.001; Figure 2A). The decrease in MVIC was greater for very old adults than young adults at stages 1, 2 and 3. At exhaustion, MVIC loss was greater for young (-28 ± 9%) compared to old adults (-19 ± 8%), but not very old adults (-23 ± 8%; Figure 2A). Db100 showed a significant effect of stage (P < 0.001; Figure 2B), indicating that the response to FNMS decreased throughout the test without agerelated differences. Significant age × stage interaction was found for VA (P = 0.027; Figure 2C), with a lower voluntary activation at stage 1 for very old adults compared to baseline and young adults.

Discussion

The present study investigated the age-related differences in performance and performance fatigability using a standardized isometric fatiguing test in men and women. Greater relative performance (number of contractions) was observed for old adults compared to young and very old adults, independent of sex. Similar total work was observed for young and old adults, with very old adults showing the lowest total work values. After the QIF test, force loss was greater for young compared to old adults without differences with very old adults, independent of sex. Impairments in contractile function were similar across ages, with no changes in voluntary activation.

Neuromuscular characteristics at baseline

Maximal force production capacity (absolute and relative to body weight) decreased with age for both sexes. This could be explained by a greater fat mass accumulation with advancing age, lowering the force-body weight ratio, or by a decrease in muscle quality (strength per unit of muscle volume).

Other than age, a decreased muscle quality could be induced by the lower physical activity of very old adults compared to the other age groups. Indeed, physical activity has a positive effect on the maintenance of muscle quality with ageing (Barbat-Artigas et al. 2014), and appears particularly important in maintaining muscle function and autonomy in very old adults (Varesco et al. 2021a).

Furthermore, the force evoked at rest by FNMS decreased with age, in agreement with previous studies evaluating the KE muscles (Senefeld et al. 2013;Mcphee et al. 2014;Dalton et al. 2015;Solianik et al. 2017), evidencing a loss of muscle contractile tissue with age. VA at baseline was similar between age groups. With age, a moderate alteration in VA was previously observed, but results are heterogeneous between studies (Rozand et al. 2020). The absence of age-related differences in VA in the present study might be due to the relatively high physical activity level of the participants (Tudor-locke et al. 2011), which has been shown to preserve the capacity to activate the working muscles (Rozand et al. 2020;Varesco et al. 2021b). It is possible, however, that the effectiveness of FNMS was limited by the fat thickness of older and very old adults (Tomazin et al. 2011), limiting the interpretation of those results despite our precautions.

Performance and performance fatigability

Old adults performed more contractions compared to the other two groups. However, because young adults were stronger than old adults, total work performed (absolute performance) was similar between those two groups. MVIC loss at exhaustion was greater for young than for old adults, but not very old adults. These results indicate greater fatigue resistance for the old adults (similar absolute performance with reduced force loss), but not very old adults (similar force loss, but lower absolute performance) compared to young adults. In older adults, the preferential loss in fast-twitch motor units is associated with an increase in slow-twitch motor units proportion and size with age, resulting in a relatively higher reliance on the oxidative metabolism (Lanza et al. 2005;Hunter et al. 2005;Krüger et al. 2018;McNeil and Rice 2018). This would lead to a greater fatigue resistance for old than young adults during isometric tasks at relative workloads (Hunter et al. 2005;Solianik et al. 2017;Krüger et al. 2018). Similar results were previously found on middle-age adults compared to young adults using a similar QIF test (Bachasson et al. 2016).

A novel finding of the present study is the loss of greater fatigue resistance in very old adults compared to young adults during isometric tasks of the KE. This agrees with previous evidences on the elbow flexors during an isometric intermittent time to exhaustion task at 60% MVIC (Allman and Rice 2001). These authors did not observed any differences in time to exhaustion or performance fatigability etiology between young and very old adults, despite the greater absolute workload in young adults (Allman and Rice 2001). In very old adults, muscle atrophy and loss of both slow-and fast-twitch motor units would decrease neuromuscular function compared to old adults (Purves-Smith et al. 2014;Hunter 2018). We reported greater force loss at low workloads (i.e. 10%, 20% and 30% of MVIC) for very old compared to young adults. At exhaustion, MVIC percentage loss of very old adults was between that of young and old adults, without significant difference from the latter two groups, as already reported for concentric tasks (Sundberg et al. 2018b).

No sex-related differences in performance fatigability were observed for the three age groups. Similar results were reported during intermittent isometric task of the plantar dorsiflexors (Kent-Braun et al.

2002

) and concentric task of the KE (Sundberg et al. 2018b). Conversely, women showed lower performance fatigability compared to men after a sustained 120-s KE MVIC independent of age (Solianik et al. 2017), probably because they performed lower total work (Solianik et al. 2017;Hureau et al. 2021). Taken together with the literature, the present results indicate that sex-related differences in performance fatigability are task-specific in young and older adults.

The significant drop in Db100 for the three age groups indicated impairments in contractile function throughout the QIF test, without age-or sex-related differences. Different results have been observed

in studies comparing young adults with old (Krüger et al. 2018) and very old (Allman and Rice 2001;Sundberg et al. 2018b) adults, probably because of the different fatiguing protocols adopted (Thomas et al. 2018). In isometric mode, blood flow occlusion induced by sustained contractions impedes oxygenated blood to reach the capillaries (Moritani et al. 1992). This facilitates impairments in contractile function (Copithorne and Rice 2020), attributed to an alteration in excitation-contraction coupling linked with high ATP turnover, free phosphate and H + accumulation (Sundberg and Fitts 2019). Metabolites accumulation in the working fibers and the recruitment of fast-twitch motor units, composed by type II fibers [less oxygen dependent (Moritani et al. 1992)], could impair performance particularly in very old adults (Sundberg et al. 2019). Regarding central impairments, high levels of physical activity of our participants might prevented an observable alteration in VA, as previously reported comparing trained and untrained individuals (Bachasson et al. 2016). Nevertheless, differences in fatigability in absence of impairments in VA have already been observed in studies investigating sex- (Hunter et al. 2006) and age-related differences (Sundberg et al. 2018b).

Conclusions

The present study showed that the greater fatigue resistance of old adults during an isometric intermittent fatiguing task of the KE was not maintained at very old age compared to young adults.

Performance fatigability at exhaustion was likely due to impairments in contractile function for the three age groups. Finally, performance fatigability and its etiology were similar between men and women independent of age.

Introduction

The inherent deterioration in muscle structure and motor command with advancing age results in a progressive loss of strength, power and physical function, which are associated with reduced autonomy and quality of life (Van Roie et al. 2011;Buckinx and Aubertin-Leheudre 2019). These age-related alterations are exacerbated at very old age (i.e. >80 years old), increasing the risk of frailty, hospitalization and ultimately death (Newman et al. 2006). While it is critical to study agerelated alterations in force production, further considering the ability to repeatedly perform muscle contractions as required during daily activities is crucial. These repeated actions are known to induce performance fatigability, i.e. decline in maximal strength or power.

The age-related alterations in strength and performance fatigability depend on the task characteristics, such as muscle groups involved or mode of contraction (Hunter et al. 2016;Hunter 2018;Krüger et al. 2018). It has been shown that the deterioration in muscle function would particularly affect the large muscle groups of the lower limbs, such as the knee extensors [START_REF] Janssen | Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr[END_REF]Hughes et al. 2001a;[START_REF] Ditroilo | Effects of age and limb dominance on upper and lower limb muscle function in healthy males and females aged 40-80 years[END_REF], whose function impacts locomotion and other activities of daily living [START_REF] Hurley | Quadriceps function, proprioceptive acuity and functional performance in healthy young, middle-aged and elderly subjects[END_REF]). In the literature, the workloads of the fatiguing tasks are usually set relatively to maximal force to account for differences in muscle strength between populations. After isometric tasks of the knee extensors, healthy old adults (60-80 years old) showed lower performance fatigability than young adults [e.g. (Callahan et al. 2009;Solianik et al. 2017;Varesco et al. 2021c)].

However, this lower performance fatigability during isometric tasks is not preserved in very old adults (Varesco et al. 2021c). Conversely, following a maximal-velocity concentric task, maximal power and force losses were larger for old compared to young adults, and even more pronounced in very old adults (Sundberg et al. 2018b).

These results suggest that the age-related differences in knee extensors performance fatigability vary across tasks. However, some of the results reported by previous studies may be explained by the lower total work (absolute performance) performed by the older adults. When young and old adults performed a similar total work during single-limb concentric and cycling tasks, Weavil et al. (2018) showed lower performance fatigability in young compared to old adults. Greater alterations in performance might occur in very old adults, especially after cycling tasks that stress the cardiovascular system to a greater extent compared to single-limb tasks [e.g. (Weavil et al. 2018)].

Thus, results obtained during cycling exercises could be more transferable to the locomotor activities of daily living. Moreover, using more ecologically valid workloads would provide further insights into the changes in neuromuscular performance with age, as already suggested when studying sexrelated differences (Hureau et al. 2021). For the lower limbs, the intensity of exercise can be set relatively to body weight, as recently done to study cancer-related fatigue (Brownstein et al. 2020).

Indeed, most of the activities of daily living, such as walking, require the ability to carry its own body weight rather than a percentage of maximal capacity.

Finally, neuromuscular alterations associated to performance fatigability with aging vary across studies, but have been mainly attributed to impairments in contractile function, with minimal reduction in voluntary activation (Krüger et al. 2018). The extend of such impairments depends primarily on the contraction mode adopted and the intensity of exercise (Sundberg et al. 2018b;Weavil et al. 2018;Krüger et al. 2020;Varesco et al. 2021c), and could vary during the fatiguing tasks (Varesco et al. 2021c). For example, our research group found greater performance fatigability along an isometric task for very old compared to young adults, but no difference at exhaustion (Varesco et al. 2021c). Further studies evaluating performance fatigability kinetic along fatiguing tasks with different contraction mode (e.g. isometric, concentric and cycling tasks) would help to understand age-related differences in neuromuscular function.

Thus, the aim of the present study was to investigate performance and performance fatigability in young, old and very old men during isometric, isokinetic concentric, and cycling fatiguing tasks with workloads set as percentage of body weight. We hypothesized lower performance with advancing age independent of the task. We also hypothesized lower fatigability in old but not very old adults

During the successive experimental sessions, participants performed intermittent incremental tests either in isometric (ISO), concentric (CON) or cycling (BIKE) mode in a randomized order. ISO and CON derived from the so-called quadriceps intermittent fatigue (QIF) test initially developed in isometric (Bachasson et al. 2013) and concentric (Varesco et al. 2021d) contraction modes, respectively. In the present study, these tests were adapted to standardize the duty-cycle and duration of the stages across the three sessions.

The first part of the session was similar across visit 2, 3 and 4. First, the stimulation intensity for peripheral nerve electrical stimulation was identified. Successively, participants performed a standardized isometric warm-up that consisted of three contractions at 30%, 50% and 70% of the perceived maximal force. Then, participants performed two maximal voluntary contractions in both isometric (MVIC) and concentric (MVCC) modes [range of motion: 60° (90°-120°, knee full extension = 120°), velocity: 120°.s -1 ]. MVCC could not be performed on the cycle-ergometer. An additional trial was performed if the difference between the peak force values was >5%. For MVIC, participants were asked to push "as hard as possible", while for the MVCC they were instructed to push "as hard and fast as possible".

After 2 min of recovery, two neuromuscular evaluations (NME) were performed, separated by 1 min of rest. NME was designed to evaluate performance fatigability and its etiology using evoked twitch at rest to assess impairments in contractile function and the interpolated twitch technique to calculate voluntary activation (Merton 1954). NME consisted of a 5-s MVIC during which a single electrical stimulation was superimposed (superimposed twitch, SIT). Then, another stimulation was delivered at rest (twitch, Tw) ~2 s after the MVIC. Visual feedback of the force (or torque) signal was displayed on a large screen positioned in front of the participants and strong verbal encouragement was provided. The fatiguing tasks (QIF tests) started at least 1 min after the second NME. A schematic representation of the experimental procedure is presented on Figure 1. Starting from the second stage, the increments were set at 5% of body weight. The target load was displayed on a large screen using a 2-Nm thick line (1Nm above -1Nm below). For ISO, participants listened to a soundtrack with the contraction-relaxation rhythm. For CON, participants were instructed to start the contraction immediately after the machine passively returned the leg to the starting position (90° knee flexion). Stages were separated by 1 min. Immediately (~2 s) at the end of each stage and at task failure, NME was performed and the rate of perceived exertion (RPE) was scored on a 6-20 Borg scale (Borg 1982). Verbal encouragement were provided. Task failure was defined as 5 consecutive contractions below the target line. After the last NME at task failure, 2 MVCC were performed.

BIKE was performed on a custom-made cycle-ergometer allowing to perform NME on the bike (Doyle- Baker et al. 2017) and consisted of 120-s stages at increasing workloads at a cadence of 37.5 rpm (75 pedal stroke) to match the duty-cycle with ISO and CON. Due to different configurations of the experimental setting, the workload was calculated differently than ISO and CON. After pilot studies, to obtain similar duration for BIKE compared to ISO and CON for very old adults, the workload was set at 0.6 W.kg -1 for the first stage and 0.3 W.kg -1 for the successive increments.

Participants were provided visual feedback of the cadence and verbal encouragement. Immediately (~2 s) after each 120-s stage and at task failure, the pedals were blocked and participants performed NME, and RPE was scored. Task failure was defined when the cadence was below 37.5 rpm for 8 s,

to match what was defined for ISO and CON.

Experimental setting

For both ISO and CON, participants were seated upright on an isokinetic dynamometer (Con-Trex MJ; CMV AG, Dubendorf, Switzerland) and were tested on the right leg. Hip and knee angles were set at 95° and 90°, respectively (180° = full extension). Extraneous movements of the upper body were minimized using belts strapped across the thorax and waist. During the first visit, the position of each participant and the alignment between the dynamometer and the right knee joint rotational axis were registered and reproduced for the next visits. For BIKE, participants seated on a validated custom-made cycle-ergometer that allowed to lock the force-measuring pedals and reproduce the same position as for the isokinetic dynamometer [for further details, see (Doyle-Baker et al. 2017)].

Briefly, when the pedals were locked, the right knee and ankle angles were set at 90°, and hip angle was approximately 100° (180° = full extension). Force was measured using instrumented pedals (Model PF1.0.0, Radlabor GmbH, Freiburg, Germany) that were located between the pedal and crank.

Torque (ISO and CON) and force (BIKE) were sampled without analogical or digital filters (16/30- (AD Instruments). Reliability and agreement for the specific ergometers and neuromuscular procedures adopted in the present study are satisfactory and can be found elsewhere [see (Maffiuletti et al. 2007;Bachasson et al. 2013;Doyle-Baker et al. 2017;Varesco et al. 2021d)].

Peripheral Nerve Electrical Stimulation

Square-wave stimulus of 2-ms duration (400 V) were delivered over the femoral nerve through a constant current stimulator (DS7R, Digitimer, Welwyn Garden City, UK). The cathode electrode (Kendall MediTrace) was placed on the inguinal triangle and the anode (50  90 mm Durastick Plus electrode; DJO Global, Vista, CA) was placed midway between the great trochanter and the lower border of the iliac crest (Piponnier et al. 2019). The stimulation intensity was progressively increased by 10 mA until torque or force and compound muscle action potential recorded through EMG (Mwave) reached a plateau. Stimulation intensity at the plateau was then increased by 30% to ensure supramaximality. Mean stimulation intensity was similar across sessions and groups and on average was 21.1 ± 6.5 mA.

Data analysis

subject factor. Generalized Estimating Equations (GEE) analyses using an autoregressive (AR-1) structure were performed on data expressed relative to baseline to investigate the effect of age on the evolution of MVIC, Tw, VA and RPE for each session. Because participants performed different numbers of stages, data were compared until the last stage performed by all participants [i.e. 3 rd stage (ISO and BIKE) or 2 nd stage (CON)] and then at exhaustion (Varesco et al. 2021a). For all ANOVAs and GEEs, when significant effects were observed, Bonferroni correction was applied to post-hoc analyses. Significance was set at P ≤ 0.05. Statistical analysis was conducted using IBM SPSS software (Version 23.0, SPSS Inc., Chicago, IL).

Results

Table 1 summarizes the participants' characteristics. Physical activity was similar across the three age groups (P = 0.209). Old adults performed better at the 6MWT (P < 0.001) and the TUG test (P = 0.001) than very old adults. Three old adults and one very old adult dropped the study due to pain during the study. Thus, eleven old adults completed ISO, 10 completed CON and 13 completed BIKE. Thirteen very old adults completed ISO and BIKE, and twelve completed CON.

Neuromuscular properties at baseline are presented in Table 2. MVIC, MVCC and Tw decreased with age, while no differences in voluntary activation were observed (ISO: P = 0.113; CON: P = 0.050; BIKE: P = 0.199). The increments of 5% of body weight for ISO corresponded to 6 ± 1%, 9 ± 3% and 12 ± 3% MVIC for young, old and very old, respectively, and was significantly different between all groups (all P < 0.05). The increments of 5% of body weight for CON corresponded to 15 ± 3%, 25 ± 9% and 31 ± 8% MVCC for young, old and very old, respectively. Young adults had lower increments (%MVCC) compared to old adults (P = 0.005) and very old adults (P < 0.001), with no difference between old and very old adults (P = 0.130).

Table 2. Neuromuscular properties of the participants at baseline

Young

Old

Very old MVIC (N)

ISO 627±169 461±98 ## 324±72 ###,$ CON 590±124 440±74 ## 306±73 ###,$ BIKE 408±89 295±46 ###
212±52 ###,$ MVIC / Body weight (N/kg) ISO 8.6±1.5 5.9±1.6 ### 4.5±0.9 ###,$ CON 8.2±1.1 5.5±1.0 ### 4.3±0.9 ###,$ BIKE 5.6±0.8 3.8±0.9 ### 3.0±0.6 ###,$ MVCC (N)

ISO 483±110 333±61 ### 241±49 ###,$ CON 485±119 334±66 ## 235±49 ###,$ Tw (N) ISO 176±44 135±29 # 106±17 ### CON 170±49 124±26 # 106±17 ### BIKE 143±29 93±25 ### 74±18 ### VA (%) ISO 95±4 97±5 92±7 CON 96±3 97±4 92±7 BIKE 96±4 97±4 93±7
MVIC: maximal voluntary isometric contraction force; MVCC: maximal voluntary concentric contraction force; Tw: resting twitch; VA: voluntary activation. # = significantly different from young adults. # =P<0.05; ## =P<0.01; ### =P<0.001. $ = significantly different from old adults. $ = P<0.05.

Performance and fatigability at the QIF tests

Performance, expressed as number of stages performed, showed a significant stage × age interaction (P < 0.001). Number of stages was lower in old and very old adults compared to young adults for the three sessions (Figure 2A). For BIKE, a significant difference was also found between old and very old adults. The loss in MVIC during the QIF test showed significant stage × age interaction for all the sessions (all P < 0.001). For ISO, MVIC decreased from baseline starting from stage 1 for old and very old adults, and from stage 2 for young adults (Figure 3A). At stage 3, the reduction in MVIC was significantly lower for the young compared to very old adults, while the opposite was found at exhaustion (Figure 3A). For CON, MVIC decreased in the three age groups starting from stage 1 (P < 0.001), without age-related differences (P = 0.079; Figure 3B). For BIKE, the decrease in MVIC was significant in old and very old adults starting from stage 1, while the decrease was significant only at exhaustion for the young adults (Figure 3C). The loss in MVCC for ISO was significant reduced at exhaustion (P < 0.001) without groups difference (P = 0.947), being -24 ± 11% for young, -23 ± 10% for old and -22 ± 11% for very old. For CON, MVCC at exhaustion was reduced for the three age groups (young: -23 ± 16%; old: -21 ± 13%; very old: -26 ± 11%; all P < 0.01). No differences between groups were found at exhaustion (all P > 0.05). A significant stage × age interaction was found for all sessions for Tw (all P < 0.001). For the three sessions, the reduction of Tw occurred earlier for old and very old adults, but the reduction at exhaustion was similar in the three age groups (Figure 4). For VA, a significant stage × age interaction was found only for BIKE (P = 0.008), with a reduced VA at exhaustion for the young adults only. No main effects were observed for ISO and CON (all P > 0.05; Figure 5).

VL Mmax showed a significant stage × age interaction for BIKE only (P < 0.001), while no significant differences across stages or age groups were found for the other sessions and for the RF and VM (all P > 0.05, data not presented). VL Mmax increased from stage 2 until exhaustion for very old adults (all P < 0.05). At exhaustion, VL Mmax was also increased for young adults (P = 0.043). VL Mmax was greater for very old compared to young adults at stages 1, 2 and 3 (all P < 0.05). Amplitude of the Mwave for VL, RF and VM is presented in Supplemental Materials 1.

Perceived exertion and heart rate

For all sessions, RPE showed significant stage × age interactions (all P < 0.001). For the three sessions, RPE was lower for young adults in the early stages, but higher at exhaustion compared to old and very old adults, except for CON for which young and old adults were not significantly different at exhaustion. RPE data are presented on supplemental material 2.

For HR, significant session (P < 0.001) and age (P < 0.001) effects were found, with no significant interaction (P = 0.493). For all age groups, ISO and CON showed significant lower values compared to BIKE (all P < 0.001), without differences between ISO and CON (P = 1.000). For the three sessions, young adults (ISO: 132 ± 21 bpm, CON: 128 ± 16 bpm; BIKE: 159 ± 21 bpm) showed significantly higher HR at exhaustion compared to old (ISO: 105 ± 14 bpm; CON: 106 ± 7 bpm; BIKE: 139 ± 17 bpm, P = 0.002) and very old adults (ISO: 94 ± 15 bpm; CON: 95 ± 23 bpm; BIKE: 114 ± 17 bpm; P < 0.001), with higher values for old compared to very old adults (P = 0.038).

Discussion

The present study aimed to investigate age-related differences in performance and fatigability during isometric, isokinetic concentric, and cycling fatiguing tasks. The load was set relative to the body weight of the participants to simulate everyday life conditions. We observed an overall better performance for young adults compared to old and very old adults for the three fatiguing tasks. Old adults performed better than very old adults in the multiarticular cycling task (BIKE) only, which was accompanied by a better performance at the functional tests (i.e. 6MWT and TUG test). For BIKE, maximal force at the last common stage decreased for the old and very old adults but not young adults.

Maximal force loss at exhaustion in ISO was greater for young compared to very old adults only, with similar performance fatigability across age groups for CON and BIKE, contrary to our hypothesis. The large decrease in Tw amplitude in the three sessions independent of age indicated that performance fatigability was mainly due to impairments in contractile function. Voluntary activation was impaired at exhaustion only for the young adults in BIKE.

Functional and neuromuscular characteristics at baseline

All participants could be defined as active based on their number of steps.day -1 (Tudor-locke et al. 2011). Old adults performed better at the 6MWT and TUG test than very old adults. Because physical activity was relatively high and similar across age groups in the present study, we assumed that these age-related differences in physical function and neuromuscular performance were due to the effect of healthy aging rather than physical inactivity (Lazarus and Harridge 2018). Maximal isometric and concentric force decreased with age, with similar values than that observed in the literature on isokinetic dynamometers (Sundberg et al. 2018b) or cycle-ergometer (Krüger et al. 2020). Evoked responses (Tw) were reduced in old and very old adults compared to young adults. This would be mainly due to loss of muscle mass and fat infiltration within the muscle contractile tissue that impede an effective contraction, reducing the maximal force and power output in older adults (Harridge and Lazarus 2017). Despite lower values with advancing age, we did not observe significant differences in the evoked responses between old and very old adults, contrary to Sundberg et al. (2018). This discrepancy was probably due to differences in physical activity of very old adults between studies (4590 ± 3606 steps.day -1 in Sundberg et al. (2018) vs. 9366 ± 3127 steps.day -1 in the present study).

Indeed, physical activity has been shown to potentially preserve muscle function and performance, as well as improve voluntary activation, even in very old adults (Barbat-Artigas et al. 2014;Rozand et al. 2020;Varesco et al. 2021b, a). This might also explain the absence of age-related differences in voluntary activation, as already suggested by a recent meta-analysis (Rozand et al. 2020).

Fatigability at similar stages performed

For the common stages performed at the isometric QIF test, performance fatigability occurred earlier in old and very old adults compared to young adults. These results were most likely due to the greater absolute load performed relatively to their maximal force production capacity, which corresponded to ~6%, ~9 % and ~12% of MVIC for young, old and very old adults, respectively. This was confirmed by the lower perceived exertion in young compared to older adults for all common stages, and lead to significant impairments in Tw starting from the first stages for old and very old adults but no impairment in voluntary activation. However, even if very old adults performed at higher absolute workload compared to old adults, no differences between those two groups were found in performance fatigability or perceived exertion for all common stages. Previously, it was proposed that the slowing in muscle contractile properties with advancing age would lead to lower MU firing rates, resulting in a reduced cost of contraction for the isometric mode (Kent-Braun 2009;Tevald et al. 2010). The lower cost of contraction in very old compared to old adults might have counterbalanced the difference in absolute workload between groups, resulting in similar performance fatigability.

For CON, no age-related differences in performance fatigability were observed at the two common stages. This may be due to the reduced number of stages performed by old and very old adults limiting the development of greater level of performance fatigability (Thomas et al. 2018), and the higher variability observed in these groups. Old and very old adults showed lower Tw compared to young adults at the last common stage. The greater workload increments in old (~25% MVCC) and very old (~31% MVCC) adults compared to young adults (~15% MVCC) probably induced a faster rise in metabolite accumulation (Sundberg et al. 2019). These results were supported by the greater perceived exertion observed for old and very old adults compared to young adults at all common stages.

For BIKE, performance fatigability and decrease in Tw occurred earlier in old and very old adults, similarly to previous observations when young and old adults were matched for total work during a cycling task (Weavil et al. 2018). The different rate of performance fatigability development observed in BIKE between young and older adults could be due to the reduction in cycling efficiency (Sacchetti et al. 2010) and anaerobic capacity, because of the preferential atrophy of fast-twitch fibers with aging and the higher reliance on the aerobic metabolism (Hunter et al. 2016;Allen et al. 2021). The decrease in maximal heart rate and cardiac output with aging (Wilson and Tanaka 2000;Poole et al. 2016;Krüger et al. 2020) could induce lower oxygen delivery to the muscles during cycling (Weavil et al. 2018) and greater metabolite accumulation inducing impairments in contractile function, as shown by the drop in Tw for old and very old. This was also supported by the greater perceived exertion for old and very old adults compared to young adults. No impairments were observed for voluntary activation in the three age groups.

Performance and performance fatigability

Isometric task. In agreement with our first hypothesis, performance was lower for old and very old adults compared to young adults during ISO with no difference between old and very old adults.

Contrary to our second hypothesis, similar performance fatigability was observed between groups at exhaustion. Previously, our research group found similar total work but lower fatigability for old compared to young adults after an isometric QIF test performed with increasing percentages of MVIC (Varesco et al. 2021c). The discrepancy in the results is likely due to differences in the workload (% body weight vs. % MVIC). Another potential explanation could be the shorter duty cycle used in the present study (0.8 s on/0.8 s off) compared to previous studies (≥ 5 s on/5 s off) (Callahan et al. 2009;Solianik et al. 2017;Varesco et al. 2021c). Indeed, sustained contractions induce greater blood flow occlusion and metabolite accumulation in young adults compared to old adults, because of their larger muscle mass and intramuscular pressure generated during isometric contractions (Russ and Kent-Braun 2003). Short contractions might prevent significant or prolonged blood flow occlusion. The lower cost of contraction in isometric mode with advancing age (Tevald et al. 2010) could partially explain the similar performance, RPE and performance fatigability in old and very old adults, despite the latter exercised at higher percentage of their MVIC.

Concentric task. In CON, old and very old adults showed lower performance compared to young adults with no difference between old and very old adults. However, performance fatigability was not different across age, contrary to our hypothesis. The age-related impairments in performance fatigability depend on total work performed (Weavil et al. 2018) but also contraction velocity (Callahan and Kent-Braun 2011;Dalton et al. 2012;Hunter et al. 2016;Senefeld et al. 2017;Sundberg et al. 2018bSundberg et al. , 2019;;Weavil et al. 2018). The results of the present study are in agreement with previous studies using similar contraction velocity (80 to 180°.s -1 ), even if the workload was set relatively to maximal power or strength (Callahan and Kent-Braun 2011;Dalton et al. 2012). Similar performance and performance fatigability were observed between old and very old adults. Previously, some evidence suggested that contraction efficiency decreased in concentric mode with advancing age (Layec et al. 2018), associated to greater metabolite accumulation (Pi, H + and dihydrogen phosphate)

within the working muscles of old adults (Sundberg et al. 2019). However, despite contraction efficiency could have influenced performance in young and older adults, this does not explain the similar performance observed between old and very old adults. For CON, the similar workload (relatively to MVCC) between old and very old adults and the high variability combined with the short duration of exercise (due to the high workload as %MVCC) could have masked some of the age-related differences between old and very old adults (Hunter et al. 2016).

Cycling task. During BIKE, we observed a decrease in performance with advancing age. This was the only session in which very old adults showed a lower performance than old adults. However, performance fatigability was similar across groups. As previously mentioned, greater involvement of the cardiovascular system for this session could explain some of the age-related differences observed in performance, and the similar performance fatigability across groups. The greater muscle mass involved during BIKE than ISO and CON, combined with decreased cycling efficiency (Sacchetti et al. 2010) and anaerobic capacity with age, could have increased the oxygen deficit. This lower oxygen availability would result in higher utilization of the anaerobic metabolism, rate of metabolite accumulation and energetic turnover, with consequent relative oxygen deficit and cardiovascular stress. This would explain the lower performance of very old adults compared to the other two groups, because this population possesses lower cardiovascular and anaerobic capacities, relying more on oxidative metabolism (Russ and Kent-Braun 2004;Varesco et al. 2021a). A rise in the cardiovascular stress combined with performance fatigability for all the muscle masses involved increased perceived exertion and induced exercise cessation for old and especially very old adults.

The reduced contribution of the cardiovascular system in ISO and CON compared to BIKE partially explain the age-related differences in performance across sessions. However, despite the similar duty cycle used in the three fatiguing tasks, the different workload used with the isokinetic dynamometer (ISO and CON) and the cycle-ergometer (BIKE) as well as the different muscle masses involved do not allow a statistical comparison of performance and performance fatigability between the three sessions. Because of its characteristics, BIKE is likely more ecologically valid than ISO and CON, and evidenced impairments in performance in very old adults compared to young and old adults for the activities of daily living, independent of physical activity.

Contractile function and voluntary activation

At exhaustion, no alterations in voluntary activation were observed in the three sessions except for young adults after BIKE. However, large impairments in contractile function were found after the three fatiguing tasks independent of age, suggesting peripheral alterations were the main responsible of performance fatigability, as previously reported in aging studies (Sundberg et al. 2018b;Krüger et al. 2018;Weavil et al. 2018). The concentrations of metabolites accumulated within the different muscles involved in cycling could be particularly important for young adults, who performed for longer duration and at higher intensities during BIKE, and could significantly activate the chemoreceptors of the groups III/IV afferents, causing impairments in voluntary activation, despite similar reductions in Tw across groups (Bigland-Ritchie and Woods 1984;Taylor et al. 2016b). For BIKE, significant alteration in the vastus lateralis M-wave amplitude were also found at exhaustion for young and very old adults. For young adults it is possible that changes in sarcolemmal excitability occurred concurrently to impairments in voluntary activation because of the great work performed.

For very old adults, lower neuromuscular junction stability with advancing age (Hepple and Rice 2016) could have played a role in altering the amplitude of M-wave despite the lower total work performed compared to the other groups. Similar impairments in contractile function at task failure for the three age groups suggest similar mechanisms or muscular limiting factor to the performance, such as actin-myosin cross-bridge formation, calcium phosphorylation, ATP depletion or drop in pH (Allen et al. 2008b, a).

Conclusion

In the present study, we showed that young men performed better than old and very old men during isometric and dynamic (single-limb and cycling) fatiguing tasks when the load was set relative to body weight.

DISCUSSION AND PERSPECTIVES

The present work aimed to evaluate the age-related changes in performance and fatigability of the knee extensors in young, old, and very old adults. In this area, studies including very old adults are scarce (Krüger et al. 2018), and the neuromuscular characteristics of this population are not well understood (Hunter et al. 2016). The age-related alterations might also be influenced by the physical activity level of the individuals (Varesco et al. 2021b). Furthermore, the age-related impairments in neuromuscular function are greater for the lower limbs compared to the upper limbs, with the knee extensors function associated with the completion of the activities of daily living, risk of falls, and frailty [START_REF] Hurley | Quadriceps function, proprioceptive acuity and functional performance in healthy young, middle-aged and elderly subjects[END_REF][START_REF] De Rekeneire | Is a fall just a fall: Correlates of falling in healthy older persons. The health, aging and body composition study[END_REF][START_REF] Pijnappels | Identification of elderly fallers by muscle strength measures[END_REF]. Finally, because most of the studies in the literature used single-limb tasks performed at intensities relative to maximal force or power (e.g. [START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF]Callahan et al. 2009;Solianik et al. 2017;Sundberg et al. 2019), and given the task-specificity of the changes in performance and fatigability with age (Hunter et al. 2016;Krüger et al. 2018), it is important to investigate the age-related changes across different contractions modes and to use more ecologically valid conditions, such as setting the workload as a percentage as body weight.

In this context, the present work included only healthy and active individuals, and we tested the knee extensors performance and fatigability across isometric and dynamic conditions, with workload set relative to maximal force production capacity and relative to body weight. To address the main objective, we separated the experimental work into three parts. The first one aimed to investigate the neuromuscular properties of very old men and women. In particular, we evaluated the association between neuromuscular, biological, and functional parameters in association with the habitual level of physical activity. The second study evaluated the differences across young, old, and very old men and women in performance, performance fatigability, and its etiology during an isometric fatiguing task, as this kind of exercise is usually adopted to compare different populations in a laboratory setting. Finally, the third study aimed to understand the age-related alterations across different fatiguing tasks and setting the workload as a percentage of body weight to simulate a more ecologically valid investigation of performance fatigability.

In the first study, we observed a significant positive association between the level of habitual physical activity and knee extensors absolute performance at the QIF test in very old women only.

Furthermore, women who possessed greater strength showed lower fat mass, greater cross-sectional area, capillarization, and enzymatic activity of fibers type I and IIA.

Firstly, we evidenced how variations in physical activity in a relatively homogeneous group of very old adults could influence important parameters such as absolute performance at the QIF test (women), performance at the time-up-and-go test (men), or even maintenance of type IIX fibers (men). Thus, we provided further evidence on the important effects of physical activity in very old adults, suggesting also that it is necessary to consider physical activity when performing aging studies (Varesco et al. 2021b).

Secondly, study 1 evidenced important sex-related differences in neuromuscular performance, biological and functional parameters. Those factors could, in theory, affect performance fatigability.

However, we found that absolute performance or performance fatigability were not related to maximal strength, structural maintenance, oxidative capacity, or enzymatic activity in both very old men and women. Indeed, in study 2, no differences were noticed between men and women in the evolution of fatigability with age, as previously observed during concentric tasks (Sundberg et al. 2018b).

Those results indicated also that, in very old adults, maximal strength of the knee extensors is not related to isometric performance and performance fatigability. Furthermore, the results obtained from study 1 also suggested that better structural maintenance, greater oxidative capacity, or higher enzymatic activity were not associated with better performance or lower performance fatigability in healthy and active very old adults. While muscle biopsies are sampled by a restricted area of a single muscle fascicle, performance fatigability is a more complex phenomenon that is determined by multiple aspects. Biological parameters obtained from muscle biopsies might represent only a small part of these factors or not well represent the whole muscle function. Thus, the association between biological parameters and performance fatigability is not obvious.

With this premise, in study 2 we wanted to investigate the changes in performance fatigability of the knee extensors in healthy active young, old, and very old men and women during a standardized isometric intermittent task. We reported similar absolute performance but lower performance fatigability for old adults compared to young and very old adults. We observed different results after the isometric fatiguing test of study 3, with a lower performance in old adults compared to young adults, and similar performance fatigability across age. This difference could be explained by the greater relative workload for old and very old adults than young adults in study 3, as it was set as a percentage of body weight. Indeed, young and older adults had a similar weight, but young adults were stronger than older adults. The greater fatigue resistance in old adults compared to young adults in study 2 may be explained by the lower blood flow occlusion and metabolite accumulation (Russ and Kent-Braun 2003) or lower cost of contraction for older adults in isometric mode (Tevald et al. 2010). In the isometric task of study 3, we used a similar protocol but with shorter contractions (0.8 s) that might prevent significant blood flow occlusion. Previously, by occluding blood flow in young and old adults when performing an isometric task of the ankle dorsiflexors, [START_REF] Chung | Age-related resistance to skeletal muscle fatigue is preserved during ischemia[END_REF] did not found a main effect of ischemia on the age-related differences. These authors excluded ischemia as the main factor explaining different fatigue resistance with aging [START_REF] Chung | Age-related resistance to skeletal muscle fatigue is preserved during ischemia[END_REF]). However, given the high density of type I muscle fibers [START_REF] Holmbäck | Structure and function of the ankle dorsiflexor muscles in young and moderately active men and women[END_REF] and the relatively small agerelated impairments in the ankle dorsiflexors function until very old age [START_REF] Mcneil | Motor unit number estimates in the tibialis anterior muscle of young, old, and very old men[END_REF]), these results might not be valid for the knee extensors and deserve further investigations.

In study 2 it was also observed that fatigue resistance during the isometric task was not maintained at very old age. We defined fatigue resistance as a lower performance fatigability associated with a greater or at least similar performance between groups. Indeed, while performance was maintained in old adults possibly because of lower performance fatigability, very old adults showed lower absolute performance and similar fatigability compared to the other two age groups. Similar results

were previously reported comparing very old and young adults after an isometric task on the upper limbs (Allman and Rice 2001). In study 2, we suggested that the important atrophy and accelerated decline in motor function could reduce the knee extensors performance and fatigability at very old age (Purves-Smith et al. 2014;Hunter 2018). It is possible that, in study 2, maximal force influenced the age-related differences in performance, as the workload was set in percentage of MVIC, while in study 3 the workload was set in percentage of body weight. In study 3, very old adults performed at higher absolute workload than old adults, because of the decreased MVIC/body weight ratio. In study 3 we observed similar performance and performance fatigability for old and very old adults after the isometric task, with significantly lower performance fatigability in very old than young adults.

Previously, it was proposed that during isometric contractions the cost of contractions would be reduced with advancing age, because of the progressive slowing in muscle contractile properties and the lower MU firing rates [START_REF] Connelly | Motor unit firing rates and contractile properties in tibialis anterior of young and old men[END_REF]Tevald et al. 2010). Although speculative, the greater economy of contraction in the isometric mode for very old adults could have partially compensated for the greater absolute workload performed in study 3 compared to old adults. However, the main cause is possibly attributable to the great variability in physiological responses typical of older adults that masked some of the age-related differences in performance and fatigability (Hunter et al. 2016).

This means that during the isometric task of study 3, variability overcame a possible difference between old and very old adults. For example, in study 1 we showed large variability across various biological and functional parameters in a homogeneous group of healthy very old adults in terms of age and physical activity, with slight changes in step count influencing both biological and functional characteristics. Taken together, the results obtained in studies 2 and 3 suggest that contraction rhythm and workload influenced the difference in performance and fatigability between old and very old adults in isometric mode. Kent-Braun 2011;Dalton et al. 2012;Sundberg et al. 2018b). This short duration of the concentric task, combined with great variability in physiological outcome with advancing age (Hunter et al. 2016), could also explain the similar performance observed between old and very old adults for the concentric task.

The cycling fatiguing task was performed to investigate performance and performance fatigability in a laboratory setting, but getting closer to the physical demands of the activities of daily living. In particular, locomotor activities usually requires the coordination and co-contraction of multiple muscle groups and sustain a load proportional to the individuals' body weight. The characteristics of the task (contraction rhythm, stage duration) were as similar as possible to the single-limb tasks, although we recognized that direct comparison across tasks was not possible. The results obtained in the cycling session showed reduced performance with advancing age but, surprisingly, performance fatigability was similar across the three age groups. We argued that the large muscle mass involved and the decreased cycling efficiency with age (Sacchetti et al. 2010) raised the rate of metabolite accumulation, energetic turnover, leading to oxygen deficit and increased, in fine, cardiovascular stress. Lower cardiovascular performance is usually observed with advancing age [START_REF] Tanaka | Age-predicted maximal heart rate revisited[END_REF] but, at the same time, old and especially very old adults rely mainly on the oxidative metabolism for muscular work (Russ and Kent-Braun 2004;Varesco et al. 2021a). This greater cardiovascular stress, combined with performance fatigability from all the muscle mass involved increased perceived exertion, leading to task failure. Theoretically, this cascade of mechanisms would have occurred first in the population who possess the lower cardiovascular and muscular performance, i.e. very old adults. This did not occurred, as old and very old showed alterations in performance fatigability at the same stages. It is possible that during the first stages, cardiovascular stress was not great enough or the large variability could masked some age-related impairments. While performance fatigability was similar across age for the knee extensors, other muscles involved in the cycling task [i.e. knee flexors, hip extensors, plantarflexors etc. [START_REF] Ericson | Power output and work in different muscle groups during ergometer cycling[END_REF]] may show a different behaviour, limiting performance.

The task was performed at a constrained rpm (37.5), which lead to the development of similar performance fatigability in the three groups, associated with similar reduction in contractile function at exhaustion. Young adults performed for a longer duration and thus reached a higher workload, leading to greater absolute metabolite accumulation but similar relative performance fatigability compared to the other groups. This large metabolite accumulation across the different muscles involved in the task could also explain the impairments in voluntary activation observed only for this group at exhaustion. Indeed, the group III/IV afferents chemoreceptors respond to great metabolite accumulation, and it has been reported as the main mechanism leading to the decrease in voluntary activation (Bigland-Ritchie and Woods 1984;Taylor et al. 2016b). Although speculative, singlelimbs intermittent tasks might not be sufficient to increase the concentration of metabolite and to induce impairments in voluntary activation in studies 2 and 3 and previous investigations (Sundberg et al. 2018b;Krüger et al. 2018), even if other studies suggest these alteration may be task-dependent (Solianik et al. 2017;Weavil et al. 2018).

CONCLUSION

Taken together, the results of the present work showed that in healthy and active individuals the agerelated differences in performance fatigability vary across contraction modes, workload selected and age groups evaluated. The association observed between physical activity level and knee extensors performance (women) or functional tests (men) suggest that walking may be a sufficient stimulus to promote adaptations in very old adults. However, differences in performance or fatigability were not directly associated with specific biological characteristics. By comparing populations of young, old and very old adults, we observed lower performance fatigability in old adults compared to young adults independent of sex after an isometric fatiguing test with a workload set relatively to maximal force production capacity. The absence of difference between young and very old adults suggests that the greater fatigue resistance of old adults is not maintained in very old adults. Finally, when the workload was set relative to individual body weight, young men showed better performance than old and very old men during isometric, concentric and cycling fatiguing tasks, and differences between old and very old men in the cycling task only. However, performance fatigability was similar between groups for the three conditions. The results of the cycling task and the specific workload used to simulate ecologically valid conditions suggest that very old adults would be more affected during activities of daily living. Finally, performance fatigability was mainly driven by impairments in contractile function independent of age, sex and contraction mode.

PERSPECTIVES

The present work offers different future perspectives on the investigation of the age-related differences between old and very old adults. Age could be used as a continuous variable to evaluate the association between age and performance fatigability. This type of analysis was not relevant in our studies because of the homogeneity in the age of our participants, especially in very old adults who were recruited from a cohort with a maximum of 3-years difference between participants.

Furthermore, our sample of healthy and active very old adults could represent the fittest part of this population, and represents only a limited portion of the general population of very old adults.

Consequently, the results observed in those individuals might not be extended to the whole population of octogenarians.

The participants of all our studies were considered as physically active based on their number of steps per day. Despite the homogeneity in age and physical activity in the very old population, we were able to observe that even slight variation in physical activity could impact neuromuscular performance (women) and other functional parameters (men) in very old adults. However, we were not able to capture the real effect of physical activity on the muscular characteristics of very old adults as we did not recruit sedentary participants. Future studies should exhaustively compare differences in physiological function between active and inactive older individuals to investigate the effects of inherent aging and those of physical inactivity.

The evaluation of old and very old adults in a laboratory setting does not represent an ecologically valid condition and the transferability of the results to the activities of daily living may be limited.

Performing fatiguing tasks with workloads set in percentage of body weight could be more representative of these activities but is not sufficient. Evaluating the association between such tests and performance at the activities of daily living along the whole day would inform on the transferability of these parameters on the real-life setting.

Finally, future studies could investigate performance fatigability after eccentric contractions in very old adults. Indeed, many activities of daily living involve eccentric actions like going down the stairs, sitting, putting down objects and controlling the speed of movement, etc.. It has been shown that eccentric strength was better preserved than isometric and concentric strength with age, but little is known about very old adults. Furthermore, eccentric exercise involves the cardiovascular system at a lower extent than concentric exercise, with a greater force developed. Thus, eccentric training could be an interesting opportunity for very old adults for which the alterations in the cardiovascular system induce early fatigue occurrence and could be a limiting factor for training efficiency.

FATIGABILITY AND NEUROMUSCULAR PERFORMANCE OF OLD AND VERY OLD HEALTHY ADULTS

ABSTRACT:

The objectives of this thesis were to evaluate neuromuscular function and performance fatigability (loss of maximal strength with fatigue) in young (<35 yr), old (>60 yr) and very old adults (>80 yr) across different fatiguing protocols. To do so, we first characterized neuromuscular function of very old adults in relationship with daily physical activity and biological parameters obtained via muscle biopsies. The results of this work showed that differences in muscle performance were sex dependent and related to the level of physical activity in very old women only. Successively, we compared performance and performance fatigability in young, old and very old adults using an intermittent isometric fatiguing task performed relatively to maximal isometric force. We observed greater fatigue resistance for old adults compared to young adults that was not maintained at very old age. No sex-related differences were observed. We finally tested the age-related differences in a more ecologically valid condition. Thus, we studied the age-related differences during an isometric, a concentric isokinetic and a cycling fatiguing task. The workload was based on the individual body weight. The results showed an overall greater performance for young adults compared to the older adults. Similar performance was observed for old and very old adults at the isometric and concentric tasks, while lower performance was observed for very old adults at the cycling task. No differences in performance fatigability were observed between old and very old for this latter session suggesting that the greater involvement of the cardiovascular system might induced very old participants to stop the exercise.

We showed that the age-related differences in performance and performance fatigability vary across tasks and when the intensity is set relative to maximal force production capacity or body weight.

FATIGUE ET PERFORMANCES NEUROMUSCULAIRES D'ADULTES SAINS ÂGÉS ET TRÈS ÂGÉS

RESUME :

Les objectifs de cette thèse étaient d'évaluer la fonction neuromusculaire et la fatigabilité (perte de la force maximale avec la fatigue) chez des personnes jeunes (<35 ans), âgées (>60 ans) très âgées (>80 ans) à travers différents protocoles de fatigue. Pour ce faire, nous avons d'abord caractérisé la fonction neuromusculaire d'adultes très âgés en relation avec l'activité physique journalière et les paramètres biologiques obtenus par biopsies musculaires. Les résultats de ce travail ont montré que les différences de performance musculaire étaient dépendantes du sexe et liées au niveau d'activité physique chez les femmes très âgées uniquement. Par la suite, nous avons comparé les performances et la fatigabilité chez des personnes jeunes, âgées et très âgées en utilisant une tâche de fatigue isométrique intermittente avec une charge relative à la force maximale isométrique. Nous avons observé une plus grande résistance à la fatigue chez les personnes âgées par rapport aux jeunes, résistance qui ne s'était pas maintenue chez les personnes très âgées. Aucune différence liée au sexe n'a été observée. L'étape suivante consistait à tester les effets de l'âge dans des conditions plus écologiques. Ainsi, nous avons étudié les différences liées à l'âge lors d'une tâche de fatigue isométrique, isocinétique concentrique et de pédalage. La charge de travail était basée sur le poids du corps. Les résultats ont montré une performance globale supérieure chez les jeunes par rapport aux personnes plus âgés. La performance était similaire chez les personnes âgées et les très âgées pour les tâches isométriques et concentriques, tandis que des performances inférieures ont été observées chez les personnes très âgés pour la tâche de pédalage. Aucune différence dans la fatigabilité n'a été observée entre les personnes âgées et très âgées pour cette dernière session, ce qui suggère la plus grande implication du système cardiovasculaire pourrait conduire les personnes très âgées à arrêter l'exercice. Nous avons montré que les différences liées à l'âge dans la performance et la fatigabilité varient selon les tâches et la façon de sélectionner l'intensité d'exercice.

  FIGURE 1. SCHEMATIC REPRESENTATION OF THE NEUROMUSCULAR SYSTEM AND THE SITES / MECHANISMS THAT COULD BE IMPAIRED DURING PHYSICAL EFFORTS. FROM: (HUNTER 2018). ............................................................................ FIGURE 2. REPRESENTATION OF THE MAIN MECHANISMS THAT CONVERT THE NERVOUS STIMULI INTO CONTRACTILE FORCE. FROM: (TORTORA AND DERRICKSON 2014). ................................................................................................. FIGURE 3. RESPONSES OBTAINED FOLLOWING ELECTRICAL OR MAGNETIC STIMULATION ON THE FORCE AND EMG CHANNEL. ................................................................................................................................................................. FIGURE 4. SCHEMATIC REPRESENTATION OF THE REAL TIME DATA ACQUISITION OF THE SILENT PERIOD THAT FOLLOWS THE MOTOR EVOKED POTENTIAL. .............................................................................................................................. FIGURE 5. TYPICAL ASSESSMENT OF VOLUNTARY ACTIVATION VIA PERIPHERAL NERVE STIMULATION ........................... FIGURE 6. ILLUSTRATION OF THE AGE-RELATED TRANSFORMATION IN THE MOTOR UNITS. ADAPTED FROM (HEPPLE AND RICE 2016)................................................................................................................................................................ FIGURE 7. CROSS-SECTIONAL VIEW OF THE MID-TIGHT IN HEALTHY YOUNG AND OLD MEN. FROM: (SERGI ET AL. 2016).. FIGURE 8. CHANGES IN THE TORQUE-VELOCITY RELATIONSHIP AND POWER-VELOCITY RELATIONSHIP WITH AGING. FROM: (RAJ ET AL. 2010). .................................................................................................................................................... FIGURE 9. A MODEL EXPLAINING THE INCREASED METABOLIC ECONOMY IN THE OLDER ADULTS MUSCLE. FROM: (KENT-BRAUN 2009). ........................................................................................................................................................... FIGURE 10. IMPAIRMENTS IN POWER PRODUCTION COULD RESULT IN FUNCTIONAL LIMITATION AND DISABILITY. FROM (REID AND FIELDING 2012). ..................................................................................................................................... FIGURE 11. EFFECT OF AGE AND PHYSICAL ACTIVITY ON MUSCLE MASS AND FAT INFILTRATION OF THE MID-THIGH. FROM: (WROBLEWSKI ET AL. 2011). .................................................................................................................................... FIGURE 12. ILLUSTRATION OF THE EXPERIMENTAL SETTING FOR THE NEUROMUSCULAR EVALUATION IN STUDY 1 AND 2 ON THE CUSTOM-MADE ISOMETRIC CHAIR................................................................................................................. FIGURE 13. ILLUSTRATION OF THE EXPERIMENTAL SETTING FOR THE NEUROMUSCULAR EVALUATION ON THE ISOKINETIC DYNAMOMETER. ....................................................................................................................................................... FIGURE 14. ILLUSTRATION OF THE EXPERIMENTAL SETTING FOR THE NEUROMUSCULAR EVALUATION ON THE CYCLO-ERGOMETER. ............................................................................................................................................................. FIGURE 15.ELECTRODE PLACEMENT OVER THE VASTUS LATERALIS, VASTUS MEDIALIS AND RECTUS FEMORIS MUSCLES .FIGURE 16. SCHEMATIC REPRESENTATION OF THE QIF TEST. ...........................................................................................

  et avec une telle équipe ! Merci aux Drs. Julien Verney, Daniel Bechet et Clément Foschia pour votre précieux aide et votre mise à disposition quand j'en avais vraiment besoin. Merci à Laurent, Gianlu et Giuseppe pour m'avoir investi dans leur projet à Besançon, pour le bel travail effectué et pour leur amitié. Merci au personnel de l'IRMIS Damien, Rodolphe, à Diana, Florianne, Severine, Floriane, Thierry, Jeremy, Jérôme, David, Jean-Benoît, Angèle, un merci aussi à Laurent Messonier pour ton animation du groupe PAF. Je voudrais aussi remercier mes copains d'aventure Marie Caroline, Clara, Pedro, Yann, Nicolas, Djahid. Je vous souhaite d'avoir tout le succès que vous méritez dans votre carrière et pour la continuation de votre thèse.

Figure 1 .

 1 Figure 1. Schematic representation of the neuromuscular system and the sites / mechanisms that could be impaired during physical efforts. From: (Hunter 2018).

Figure 2 .

 2 Figure 2. Representation of the main mechanisms that convert the nervous stimuli into contractile force. From: (Tortora and Derrickson 2014).

Figure 3 .

 3 Figure 3. Responses obtained following electrical or magnetic stimulation on the force (left) and EMG channel (compound muscle action potential, right).

Figure 4 .

 4 Figure 4. Schematic representation of the real time data acquisition of the silent period (SP) that follows the motor evoked potential (MEP).

À

  RETENIR: NON-INVASIVE MEASUREMENT OF PERFROMANCE FATIGABILITY L'évaluation et l'interprétation de la fatigabilité et des changements dans le système neuromusculaire nécessitent une méthodologie claire et acceptée. Les techniques de stimulation couplées à des mesures de force et de signal électromyographique permettent de quantifier les altérations de la fonction contractile et/ou de l'activation volontaire de manière non-invasive. La technique de la secousse interpolée est la procédure la plus utilisée pour évaluer l'étiologie de la fatigabilité. Les stimulations peuvent être délivrées au niveau du cortex moteur (magnétique), du nerf moteur (magnétique et électrique) ou sur le ventre musculaire (électrique). Pour terminer, la durée et l'intensité de stimulation peuvent être adaptés à la population et au but de l'étude.

  and type IIx fibers by slow-twitch MU.These phenomenon associated with aging radically change some of the typical characteristics of MU observed in young men. First, fibers with different myosin-heavy-chain expression can be observed into the same MU, with the newly-acquired fibers adapting to the characteristics of the MU(Hepple and Rice 2016; McNeil and Rice 2018). Second, because axonal sprouting and reinnervation are limited to a small compartment of the muscle, MU fibers are clustered in a limited area [Figure6;(Hepple and Rice 2016)].

Figure 6 .

 6 Figure 6. Illustration of the age-related transformation in the motor units. Adapted from (Hepple and Rice 2016).

Figure 7 .

 7 Figure 7. Cross-sectional view of the mid-tight in healthy young (25 years old, left) and old (65 years old, right) men. From: (Sergi et al. 2016).

  adaptation to slowing of the muscle contractile properties with aging (Kent-Braun 2009; Tevald et al. 2010). Because of the important deterioration of MU with advancing age, these results might change at very old age. Studies investigating voluntary activation in very old adults are limited in the literature, and do not allow a clear information on whether deficits in voluntary activation are greater in very old adults (>80 years old) than old adults (60-79 years old) (Rozand et al. 2020). Physical activity could play a role in the maintenance of voluntary activation in older adults. For instance, studies investigating the effects of resistance training in older individuals found improved voluntary activation and decreased inhibition (Christie and Kamen 2010), accompanied by greater MU firing

Figure 8 .

 8 Figure 8. Changes in the torque-velocity relationship (1) and power-velocity relationship (2) with aging (dotted line). OA: old adults; YA: young adults; Pmax: Maximal Power; Fmax: Maximal force; Vmax: Maximal velocity. From: (Raj et al. 2010).

Figure 9 .

 9 Figure 9. A model explaining the increased metabolic economy in the older adults muscle. From: (Kent-Braun 2009).

Figure 10 .

 10 Figure 10. Impairments in power production could result in functional limitation and disability. From (Reid and Fielding 2012).

À

  RETENIR: AGE-RELATED ALTERATIONS IN FATIGABILITYLes différences de fatigue avec l'âge varient en fonction des tâches effectuées. Les personnes âgées montrent une moindre fatigabilité après des tâches de fatigue isométriques. Ces différences sont atténuées lors de tâches intermittentes qui réduisent les effets négatifs de l'occlusion du flux sanguin.Lors de contractions isocinétiques et isotoniques, la performance et la fatigabilité diminuent avec l'âge, avec des altérations plus importantes à des vitesses de contraction plus rapides. Enfin, lors de tâches multiarticulaires comme le cyclisme, les détériorations de performance et de fatigabilité avec l'âge dépendent de la durée et de l'intensité de l'effort, le système cardiovasculaire contribuant largement à ces différences liées à l'âge lors d'efforts intenses et prolongés.5. EFFECTS OF PHYSICAL ACTIVITY ON NEUROMUSCULAR FUNCTIONPhysical activity level could influence the individual rate of muscle deconditioning with advancing age[START_REF] Bouchard | Physical Activity, Fitness, and Health : International Proceedings and Consensus Statement[END_REF]. Aging is associated with a reduction in quantity and intensity of physical activity level(Tudor-locke et al. 2011;[START_REF] Amireault | Physical Activity Preferences Among Older Adults: A Systematic Review[END_REF] Varesco et al. 2021b). For instance, while younger adults perform a variety of exercises or sports, physical activity at very old adults mainly consists in walking[START_REF] Amireault | Physical Activity Preferences Among Older Adults: A Systematic Review[END_REF]. Both cross-sectional and longitudinal studies assessing the effects of physical activity on aging highlight the gap in physiological function and physical performance between active and inactive older adults(Varesco et al. 2021b). For instance, muscle quality (strength per unit of muscle volume) has been shown to be preserved with age in active individuals from 50 to 80 years old, but decreases in inactive older adults(Barbat-Artigas et al. 2014).Physical activity could also play a role in attenuating the age-related decline in corticospinal excitability[START_REF] Hassanlouei | Physical activity modulates corticospinal excitability of the lower limb in young and old adults[END_REF][START_REF] Rozand | Differential effects of aging and physical activity on corticospinal excitability of upper and lower limb muscles[END_REF], skeletal muscle lipid content (St-Jean-Pelletier et al. 2017) and skeletal muscle mitochondrial density[START_REF] Distefano | Physical activity unveils the relationship between mitochondrial energetics, muscle quality, and physical function in older adults[END_REF]. A large body of studies investigated the physiological characteristics of master athletes as a model of inherent or healthy aging[START_REF] Lazarus | The relationships and interactions between age, exercise and physiological function[END_REF], without the negative effects of physical inactivity. The analyses of performance during endurance competitions showed that performance decreases by ~5 to 20% per decade starting from ~40 years old, arguing that this rate of decline represents the effect of age only[START_REF] Hoffman | The western states 100-mile endurance run: Participation and performance trends[END_REF][START_REF] Hoffmann | Performance Trends in 161-km Ultramarathons[END_REF][START_REF] Knechtle | Age-related changes in 100-km ultramarathon running performance[END_REF][START_REF] Lazarus | Declining performance of master athletes: silhouettes of the trajectory of healthy human ageing[END_REF] Varesco et al. 2019b). Lifelong endurance exercisers showed greater maximal aerobic capacity, accompanied by greater left ventricular mass, muscle capillarization and aerobic enzyme activity compared to inactive older adults[START_REF] Gries | Cardiovascular and skeletal muscle health with lifelong exercise[END_REF][START_REF] Carrick-Ranson | The effect of lifelong endurance exercise on cardiovascular structure and exercise function in women[END_REF]). The master athletes showed preserved muscle mass, reduced fat infiltration [e.g. Figure11;[START_REF] Wroblewski | Chronic Exercise Preserves Lean Muscle Mass in Masters Athletes[END_REF]], higher neuromuscular transmission stability(Power et al. 2016a) and greater power development per type I muscle fiber compared to inactive older adults[START_REF] Gries | Single-muscle fiber contractile properties in lifelong aerobic exercising women[END_REF]).

Figure 11 .

 11 Figure 11. Effect of age and physical activity on muscle mass and fat infiltration of the mid-thigh. From: (Wroblewski et al. 2011).

  TAKE HOME MESSAGE: EFFECTS OF PHYSICAL ACTIVITY ON NEUROMUSCULAR FUNCTION Physical activity preserves or attenuates the age-related decline of neuromuscular and cardiovascular function. Because active and inactive older adults show different characteristics, physical activity should be checked when performing aging studies. Active and inactive participants should be separated to reduce the between-subjects variability and differentiate the effects of aging and physical inactivity. À RETENIR: THE EFFECTS OF PHYSICAL ACTIVITY ON NEUROMUSCULAR FUNCTION L'activité physique préserve ou atténue le déclin des fonctions neuromusculaire et cardiovasculaire avec l'âge. Étant donné que les personnes âgées actives et inactives présentent des caractéristiques différentes, le niveau d''activité physique doit être vérifié lors des études sur le vieillissement. Les participants actifs et inactifs doivent être séparés pour réduire la variabilité interindividuelle et différentier les effets du vieillissement de ceux de l'inactivité physique.

Study 2 1

 21 aimed to investigate performance fatigability of the knee extensor muscles induced by the isometric quadriceps intermittent fatiguing test in healthy and active young, old and very old men and women. The workload was set relative to maximal force production capacity. It was hypothesized that young and very old adults would present similar fatigability after the isometric intermittent fatiguing test, while older adults would show a lower performance fatigability, according to previous evidence on other muscle groups(Allman and Rice 2001; Kent-Braun et al. 2002;[START_REF] Lanza | Age-related enhancement of fatigue resistance is evident in men during both isometric and dynamic tasks[END_REF].It was further hypothesized that old adults would show lower impairments in contractile function than young and very old adults, without age-related differences in voluntary activation impairments. No effect of sex on these age-related impairments in performance fatigability or etiology was expected.Study 3 aimed to compare performance and fatigability in healthy and active young, old and very old men during isometric, isokinetic concentric, and cycling fatiguing tasks. For this study, workload was set as percentage of body weight. It was hypothesized that young adults would perform better than old and very old men independent of the task. It was also hypothesized that old, but not very old men, would present lower fatigability during the isometric fatiguing task only. Greater fatigability in old and very old adults compared to young adults was expected during the concentric and cycling fatiguing tasks. It was expected that performance fatigability would be mainly driven by impairments in contractile function for all the tasks independent of age, with no alteration in voluntary activation. Isometric custom-made chair For the neuromuscular evaluation of the knee extensors (study 1 and 2), participants seated upright on a custom-build chair, as presented in Figure12. The position of the force transducer and the seat were adjusted so the knee angle was at 90° (180°= full extension). The hip angle was set at 120° to facilitate coil placement over the femoral triangle. Extraneous movements of the upper body were minimized using belts across the waist and the thorax. The force signal was acquired using a force transducer (Omega Engineering Inc., Stamford, CT, USA) firmly attached over the malleoli, and digitized at a sampling rate of 2 kHz by PowerLab System (16/30-ML880/P, AD Instruments, Bella Vista, Australia).

Figure 12 .

 12 Figure 12. Illustration of the experimental setting for the neuromuscular evaluation in study 1 and 2 on the custom-made isometric chair.

Figure 13 .

 13 Figure 13. Illustration of the experimental setting for the neuromuscular evaluation on the isokinetic dynamometer.

Figure 14 .

 14 Figure 14. Illustration of the experimental setting for the neuromuscular evaluation on the cycloergometer.

Figure 15 .

 15 Figure 15.Electrode placement over the vastus lateralis (red), vastus medialis (pink) and rectus femoris muscles (green).

1. 3 . 2

 32 Peripheral Nerve Electrical StimulationSquare-wave stimulus of 2-ms duration (400 V) were delivered over the femoral nerve through a constant current stimulator (DS7R, Digitimer, Welwyn Garden City, UK) (study 3). The cathode electrode (Kendall MediTrace) was placed on the inguinal triangle and the anode (50 × 90 mm Durastick Plus electrode; DJO Global, Vista, CA) was placed midway between the greater trochanter and the lower border of the iliac crest(Piponnier et al. 2019). The stimulation intensity was progressively increased by 10 mA until the torque and EMG (M-wave) responses reached a plateau.

Figure 16 .

 16 Figure 16. Schematic representation of the QIF test. EV: evaluation of maximal force and response to stimulation. MVIC: maximal voluntary isometric contraction force.

  par jour étaient corrélés avec le travail total uniquement chez les femmes (r = 0.73, P = 0.011). Chez les hommes, le nombre de pas par jour était corrélé avec le pourcentage (r = 0,57, P = 0.033), le facteur de forme (r = 0,75, P = 0,002) et la tortuosité capillaire des fibres de type IIX (r = 0.59, P = 0.035). Aucune autre corrélation pertinente n'a été observée chez les hommes ou les femmes entre le nombre de pas par jour et les paramètres biologiques. Le niveau d'activité physique était positivement associé à la capacité des femmes très âgées à réaliser un test de fatigue, mais pas à la capacité de production de force maximale des KE. L'activité physique des hommes très âgés n'était pas corrélée à la performance musculaire. Nous suggérons que les femmes très âgées pourraient présenter un risque plus élevé de perte d'autonomie et que l'augmentation du nombre de pas par jour pourrait fournir un stimulus suffisant pour engendrer des adaptations chez les femmes moins actives.

(

  MHC-I) (A4.951 from Enzo Life Sciences, Farmingdale, NY, United States) or MHC-IIA (N2.261, Enzo life Sciences), co-labeled with anti-laminin-α1 (Sigma) to outline the fibers, and resolved with corresponding secondary antibodies conjugated to Alexa-Fluor 488 or 546 (Invitrogen). Images were captured with a high-resolution cooled digital DP-72 camera coupled to a BX-51 microscope (Olympus Life Science, Munich, Germany) at a resolution of 0.64 µm/pixel. Four fields, each containing 80 ± 15 fibers, were analyzed per subject. The contractile Type (I, I-IIA, IIA, IIA-IIX or IIX), cross-sectional area (CSA) and perimeter were determined for each fiber using Visilog-6.9 (Noesis, Gif-sur-Yvette, France) according to(Meunier et al. 2010). Thresholding of MHC images distinguished either positive, negative, or intermediate MHC labeling. Type I fibers were positive only for MHC-I, and type IIA fibers positive only for MHC-IIa. Type X fibers were negative for both MHC-I and MHC-IIa. Type I-IIA hybrid fibers were intermediate for both MHC-I and MHCIIa, while type IIA-IIX hybrid fibers were only intermediate for MHC-IIa labeling. A shape factor

  de cette étude était d'examiner les différences liées à l'âge dans la fatigabilité induite par un test de fatigue isométrique intermittent du quadriceps chez des hommes et des femmes jeunes (<35 ans), âgés (>60 ans) et très âgés (>80 ans). La perte de force maximale, la fonction contractile et le niveau d'activation volontaire des muscles extenseurs du genou ont été évalués tout au long d'un test de fatigue isométrique à l'aide de stimulations magnétiques du nerf fémoral. Les personnes âgées ont effectué plus de contractions (indice de performance relative) que les jeunes (P = 0.046) et les personnes très âgées (P = 0.007), sans différence entre les personnes jeunes et très âgées. Le travail total (performance absolue) était plus important chez les jeunes et les personnes âgées par rapport aux personnes très âgées (P < 0.001), sans différence entre les personnes jeunes et âgées. À l'épuisement, la perte de force était plus importante chez les jeunes (-28 ± 9 %) que chez les personnes âgées (-19 ± 8 %), mais pas chez les personnes très âgées (-23 ± 8 %). La réponse à la stimulation du nerf fémoral a diminué de façon similaire à l'épuisement pour les trois groupes d'âge, indiquant une altération similaire de la fonction contractile avec l'âge. Aucune altération de l'activation volontaire n'a été observée. Les altérations des paramètres neuromusculaires étaient similaires chez les hommes et les femmes. Cette étude a montré que les personnes âgées étaient moins fatigables que les jeunes lors d'une tâche de fatigue isométrique intermittente des extenseurs du genou. Cette plus grande résistance à la fatigue n'était pas maintenue chez les personnes très âgées, indépendamment du sexe. La fatigabilité à l'épuisement était principalement due à des altérations de la fonction contractile pour les trois groupes d'âge.

Figure 2 .

 2 Figure 2. Maximal isometric force (MVIC, Panel A), doublet amplitude (Db100, Panel B) and

References 3 .

 3 STUDY 3 Effects of old and very old age on performance and fatigability during isometric, concentric and cycling fatiguing tasks in men Effects of old and very old age on performance and fatigability during isometric, concentric and cycling fatiguing tasks in menGiorgio Varesco; Eric Luneau; Guillaume Millet; Léonard Féasson; Thomas Lapole; Vianney Rozand To submit This version of the paper has not been reviewed by all co-authors Résumé: L'objectif de cette étude était d'étudier la performance et la fatigabilité lors de tâches de fatigue isométrique, concentrique et de cyclisme chez des hommes jeunes, âgés et très âgés. Quinze hommes jeunes, 13 hommes âgés et 12 hommes très âgés ont effectué de manière aléatoire trois tâches de fatigue : un test incrémental de fatigue isométrique (ISO) et concentrique (CON) sur un dynamomètre isocinétique et un test similaire sur un cyclo-ergomètre (BIKE). L'ISO et le CON consistaient en des paliers incrémentaux de 75 contractions (0.8 s on/0.8 s off), avec une durée et un cycle d'utilisation similaires pour le BIKE (120 s à 37.5 rpm). Les augmentations étaient spécifiques à chaque appareil et définies en pourcentage du poids de corps. La stimulation électrique nerveuse percutanée a été utilisée pour évaluer l'activation volontaire (VA) et l'amplitude de la secousse musculaire au repos (Tw). La performance (nombre de paliers) était plus importante chez les jeunes adultes que chez les personnes âgées et très âgées pour les trois sessions, et plus importante chez les personnes âgées que très âgées pour le BIKE uniquement (P<0.001). La diminution de la force maximale (fatigabilité) était plus importante chez les jeunes adultes que chez les personnes très âgées après ISO (P<0.001), mais similaire dans tous les groupes d'âge après CON et BIKE. L'amplitude du Tw a diminué dans les trois sessions indépendamment de l'âge (P<0.001). La VA a diminué uniquement chez les jeunes adultes après BIKE (P<0.05). Ces résultats montrent que la performance diminue avec l'âge indépendamment de la tâche lorsque la charge est fixée par rapport au poids du corps individuel. La fatigabilité était plus importante chez les jeunes adultes que chez les personnes très âgées pendant la tâche isométrique, mais similaire entre les groupes d'âge pendant les tâches dynamiques. La fatigabilité était principalement due à des altérations de la fonction contractile.

Figure 1 .

 1 Figure 1. Experimental procedures for the three body-weight based quadriceps intermittent fatigue

Figure 2 .

 2 Figure 2. Number of stages performed by the young, old and very old adults at the isometric (ISO),

Figure 3 .

 3 Figure 3. Maximal voluntary isometric contraction force (MVIC) of the three age groups across the

Figure 4 .

 4 Figure 4. Twitch amplitude (Tw) of the three age groups across the isometric (ISO; panel A),

Figure 5 .

 5 Figure 5. Voluntary activation (VA) of the three age groups across the isometric (ISO; panel A),

  Rate of perceived exertion of the three age groups across the isometric (ISO; panel A), concentric (CON; panel B) and cycling (BIKE; panel C) body weight based QIF test. The last common stage was stage 3 for ISO and BIKE and stage 2 for CON. *=significant difference from baseline (P < 0.05). #=significant difference from the young adults (P < 0.05). $=significant difference from the old adults (P < 0.05).

  aux jeunes adultes pour les trois tâches effectuées. Seule l'exercice sur vélo a induit une différence de performance entre les personnes âgées et les personnes très âgées, ces dernières présentant des performances inférieures. Pour les trois tâches effectuées, la fatigue induite était similaire quel que soit l'âge. La différence de résultat entre les études 2 et 3 indiquent que la réalisation d'une tâche avec une charge relative à la force maximale plutôt qu'au poids de corps des individus pourrait masquer certaines altérations liées à l'âge dans les activités de la vie quotidienne. Étant donné que nous avons montré une détérioration croissante liée à l'âge dans des conditions plus proches des activités de la vie quotidienne (exercice sur vélo effectué en pourcentage du poids corporel), les études futures devraient envisager d'étudier les altérations neuromusculaires avec le vieillissement dans des conditions plus écologiques.

  FIGURES FIGURE 1. SCHEMATIC REPRESENTATION OF THE NEUROMUSCULAR SYSTEM AND THE SITES / MECHANISMS THAT COULD BE IMPAIRED DURING PHYSICAL EFFORTS. FROM: (HUNTER 2018). ............................................................................ FIGURE 2. REPRESENTATION OF THE MAIN MECHANISMS THAT CONVERT THE NERVOUS STIMULI INTO CONTRACTILE FORCE. FROM: (TORTORA AND DERRICKSON 2014). ................................................................................................. FIGURE 3. RESPONSES OBTAINED FOLLOWING ELECTRICAL OR MAGNETIC STIMULATION ON THE FORCE AND EMG CHANNEL. ................................................................................................................................................................. FIGURE 4. SCHEMATIC REPRESENTATION OF THE REAL TIME DATA ACQUISITION OF THE SILENT PERIOD THAT FOLLOWS THE MOTOR EVOKED POTENTIAL. .............................................................................................................................. FIGURE 5. TYPICAL ASSESSMENT OF VOLUNTARY ACTIVATION VIA PERIPHERAL NERVE STIMULATION ........................... FIGURE 6. ILLUSTRATION OF THE AGE-RELATED TRANSFORMATION IN THE MOTOR UNITS. ADAPTED FROM (HEPPLE AND RICE 2016)................................................................................................................................................................ FIGURE 7. CROSS-SECTIONAL VIEW OF THE MID-TIGHT IN HEALTHY YOUNG AND OLD MEN. FROM: (SERGI ET AL. 2016).. FIGURE 8. CHANGES IN THE TORQUE-VELOCITY RELATIONSHIP AND POWER-VELOCITY RELATIONSHIP WITH AGING. FROM: (RAJ ET AL. 2010). .................................................................................................................................................... FIGURE 9. A MODEL EXPLAINING THE INCREASED METABOLIC ECONOMY IN THE OLDER ADULTS MUSCLE. FROM: (KENT-BRAUN 2009). ........................................................................................................................................................... FIGURE 10. IMPAIRMENTS IN POWER PRODUCTION COULD RESULT IN FUNCTIONAL LIMITATION AND DISABILITY. FROM (REID AND FIELDING 2012). ..................................................................................................................................... FIGURE 11. EFFECT OF AGE AND PHYSICAL ACTIVITY ON MUSCLE MASS AND FAT INFILTRATION OF THE MID-THIGH. FROM: (WROBLEWSKI ET AL. 2011). .................................................................................................................................... FIGURE 12. ILLUSTRATION OF THE EXPERIMENTAL SETTING FOR THE NEUROMUSCULAR EVALUATION IN STUDY

  d'abord, je voudrais remercier le Pr. Stéphane Baudry et le Pr. Raphael Zory d'avoir accepté d'expertiser ce travail et de participer à ce jury de thèse. I'd like to thank Dr. Marine Gueugneau, Pr. Guillaume Millet and Dr. Christopher Sundberg for accepting to be part of the committee and appraise this work. Je voudrais remercier particulièrement mes directeurs de thèse Vianney Rozand, Thomas Lapole et Léonard Féasson. Merci de m'avoir recruté pour cette thèse et m'avoir accompagné dans cette aventure. Je vous remercie de m'avoir fait confiance, de votre disponibilité 24h/24, 7j/7 et du soutien que vous m'avez apporté du point de vue professionnel et personnel. Le bon travail d'équipe qu'on a

	REMERCIEMENTS / ACKNOWLEDGEMENTS / RINGRAZIAMENTI
	EMG	Electromyography
	CNS	Central nervous system
	SR	Sarcoplasmic Reticulum
	SERCA	Sarco-Endoplasmic Reticulum Calcium ATPase
	MVIC	Maximal Voluntary Isometric Contraction
	PNS	Peripheral Nerve Stimulation
	CMAP	Compound Muscle Action Potential
	MEP	Motor Evoked Potential
	CMEP	Cervicomedullary Motor Evoked Potential
	TMEP	Thoracic Motor Evoked Potential
	PNMS	Peripheral Nerve Magnetic Stimulation
	TMS	Transcranial Magnetic Stimulation
	SP	Silent Period
	GABAB	Gamma-Aminobutyric Acid
	PAP	Post-Activation Potentiation
	ITT	Interpolated Twitch Technique
	SIT	Superimposed Twitch
	POT	Potentiated Resting Twitch
	ERT	Estimated Resting Twitch
	MU	Motor Unit

Tout

Table 1 .

 1 Muscle fibers type characteristics

		Type I fibre	Type IIa fibre	Type IIx fibre
	Diameter	+	++	+++
	Motoneuron size	+	+++	+++
	Nerve conduction speed	+	+++	+++
	Contractile speed	+	++	++
	Motor unit strength	+	++	++
	Glycolitic capacity	+	++	++
	Fatigue resistance	+++	++	+
	Oxydative capacity	+++	++	+
	Capillarity	+++	++	+
	Myoglobin content	+++	++	+

  voluntary activation capacity seems only moderately impacted by age. These age-related impairments are dependent on the muscle group, contraction mode and velocity, . Finally, interindividual variability increases with age, making challenging to identify some of the age-related alterations in neuromuscular function.

À RETENIR: NEUROMUSCULAR SYSTEM CHANGES WITH AGING

Le vieillissement induit des altérations à la fois structurelles et fonctionnelles du système neuromusculaire, incluant le remodelage et la perte d'unités motrices. Cependant, les altérations de la capacité à développer la force et la puissance sont encore plus importantes, surtout chez les personnes très âgées. Malgré les altérations de la capacité à développer rapidement la force chez les personnes âgées, qui pourraient être dues à des altérations de la commande motrice du muscle, la capacité d'activation volontaire maximale ne semble que modérément affectée par le vieillissement. Ces altérations liées à l'âge varient en fonction du groupe musculaire, du mode et de la vitesse de contraction. Enfin, la variabilité interindividuelle augmente avec l'âge, ce qui rend difficile l'identification de certaines altérations de la fonction neuromusculaire avec l'âge.

  DATA Additional data on muscle biopsies of the vastus lateralis were obtained from a collaboration with the Myology Unit of the University Hospital of Saint-Etienne, and the National Institute of Research for Agriculture, Nutrition and Environment (INRAE) of the University of Clermont Auvergne. We

	Résumé :
	L'objectif de cette étude était d'évaluer l'association entre l'activité physique, la performance des
	extenseurs du genou (KE) (force isométrique et fatigabilité) et les paramètres biologiques (propriétés
	structurelles, microvasculaires et métaboliques du muscle) chez des hommes et des femmes très âgés
	en bonne santé. Trente adultes très âgés (82 ± 1 ans, 15 femmes) ont effectué un test de fatigue
	isométrique intermittent du quadriceps (QIF test) afin d'évaluer la force maximale des KE, le travail
	total (indice de performance absolue) et la fatigabilité. Des biopsies musculaires du muscle vaste
	latéral ont été effectuées pour évaluer la typologie et la morphologie des fibres musculaires, la
	used those data that were already analyzed to compare muscle biological properties with the
	performance (e.g. maximal force, QIF test performance, etc.) and functional (e.g. 6-MWT, TUG test
	and physical activity) data that we measured on the same participants for study 1.

microvasculature et l'activité enzymatique. Des analyses de corrélation ont été utilisées pour étudier les relations entre l'activité physique (pas/jour, actimétrie), la performance des KE et les données biologiques pour chaque sexe séparément. Les hommes, comparés aux femmes, ont montré un travail total plus important lors du QIF test (44 497 ± 8 629 Ns vs. 26 946 ± 4 707 Ns ; P < .001). Les pas

Table 1 .

 1 Characteristics of the participants

YM: young men; YW: young women; OM: old men; OW: old women; VOM: very old men; VOW: very old women; 6MWT: 6-minutes Walk Test. # = significantly different from men of the same age group. ## =P<0.01; ### =P<0.001. *= significantly different from the young group of the same sex. ***=P<0.001. $ = significantly different from the old group of the same sex. $$ = P<0.01; $$$ =P<0.001. Men were stronger than women for all age groups. MVIC decreased with age (sex × age interaction:

Table 2 .

 2 Neuromuscular characteristics of participants at baseline.

	YM: young men; YW: young women; OM: old men; OW: old women; VOM: very old men; VOW:
	very old women; MVIC: maximal voluntary isometric contraction force; BW: body weight; Db100:

amplitude of the doublet evoked at rest. VA: voluntary activation. # = significantly different from men of the same age group. ## =P<0.01; ### =P<0.001. *= significantly different from the young group of the same sex. **= P<0.01; ***=P<0.001. $ = significantly different from the old group of the same sex. $ = P<0.05; $$$ =P<0.001.

  ML880/P, ADInstruments, Bella Vista, Australia). Electromyographic (EMG) activity was recorded from the vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM) muscles of the right leg. Pairs of self-adhesive Ag/AgCl surface electrodes (recording diameter=10 mm, Kendall MediTrace 130 foam electrode, MA) were placed in bipolar configuration over the muscle belly following the SENIAM guidelines. The interelectrode (center to center) distance was 30 mm. A reference electrode was placed on the right patella. Finally, two electrodes were placed over the right clavicle and the tenth left rib, respectively, to measure hearth rate (HR). The skin was shaved, abraded, and cleaned with alcohol swab before electrode placement. EMG signals were amplified

	(gain = 500) using an octal bio-amplifier (ML138, AD Instruments) with a bandwidth frequency
	range of 10-500 Hz and digitized by a Power-Lab System (16/30-ML880/P, AD Instruments). Torque
	(or force) and EMG signals were recorded simultaneously at 2 kHz on a computer using Labchart v8

Table 1 .

 1 Characteristics of the participants

		Young	Old	Very old
	Age (years)	24±3	70±4 ###	86±1 ###, $$$
	Weight (kg)	72.1±8.8	79.2±9.5	71.0±5.0
	Height (cm)	178.0±6.9	173.8±5.4	164.7±3.7 ###,$$
	Steps.day -1	10,477±2,818	11,647±2,399	9,366±3,127
	6MWT (m)		669.7±97.9	452.1±92.2 $$$
	TUG test (s)		4.1±0.7	6.3±1.9 $$
	6MWT: 6-minutes Walk Test. TUG test: Timed Up and Go test. # = significantly different from young
	adults. ## =P<0.01; ### =P<0.001. $ = significantly different from the old group. $$ = P<0.01;
	$$$ =P<0.001.

  Old and very old adults performed similarly at ISO and CON. During the cycling task, very old adults were less performant than old adults. However, performance fatigability, i.e. loss in maximal force, was greater in young adults than very old adults during the isometric fatiguing task, but similar between age groups during dynamic and cycling tasks. Performance fatigability was mainly driven by impairments in contractile function independent of age, with only young adults showing modest impairments in voluntary activation following the cycling task.

	SUPPLEMENTAL MATERIAL 1
	M-wave amplitude (Mmax) at the isometric (ISO), concentric (CON) and cycling (BIKE) quadriceps
	intermittent fatigue test for the three muscles evaluated: the vastus lateralis (VL; panels A, B, C),
	vastus medialis (VM; panels D, E, F) and rectus femoris (RF; G, H, I). *=significant difference from
	baseline (P < 0.05). #=significant difference from the young adults (P < 0.05).

EPIGRAPH

So, now you're a Professor? Grandma The cycle ergometer (study 3) was built based on the validated one (Doyle- Baker et al., 2017). It consisted in a custom-made recumbent bike with electromagnetically-braked smart trainer (CycleOps H3, Saris, Madison, WI) working with Rouvy software (VirtualTraining s.r.o., Vimperk, Czech Republic), as presented in Figure 14. The smart trainer measured power, cadence, and speed. A mechanical block was built so the pedals could be locked instantaneously in horizontal position.

Participants were positioned on the ergometer and the seat was adjusted so the hip and knee angles were at 95° and 90°, respectively (180° = full extension) when the pedals were blocked. Extraneous movements of the upper body were minimized using belts strapped across the waist and the thorax.

A custom-made unlockable system for the ankle was used to facilitate the participants to maintain an ankle angle of 90° and reduce extraneous movements of the articulation. Participants were also instructed to maintain the ankle angle at 90° and push their foot forward. To start cycling, the anklelock system was first released, and then the pedals were unlocked (≤ 5-s). Isometric force was measured using validated wireless force transducer installed on the pedals (PowerForce pedals, Radlabor GmbH, Freiburg, Germany). Force signal was transmitted at 500 Hz to a PowerLab System (16/30-ML880/P, AD Instruments, Bella Vista, Australia).

Résumé des méthodes

Abstract (250 words)

The aim of the present study was to investigate age-related differences in fatigability induced by an isometric quadriceps intermittent fatiguing test in young (<35 years old), old (>60 years old) and very old (>80 years old) men and women. Maximal force loss, contractile function and voluntary activation of the knee extensors were evaluated throughout an isometric fatiguing test using femoral nerve magnetic stimulations. Older adults performed more contractions (index of relative performance) than young (P = 0.046) and very old adults (P = 0.007), without differences between young and very old adults. Total work (absolute performance) was greater for young and old adults compared to very old adults (P < 0.001), without differences between young and old adults. At exhaustion, force loss was greater for young (-28 ± 9%) compared to old adults (-19 ± 8%), but not very old adults (-23 ± 8%).

The response to femoral nerve stimulation decreased similarly at exhaustion for the three age groups, indicating similar alteration in contractile function with age. No impairment in voluntary activation was observed. Impairments in neuromuscular parameters were similar for men and women. This study showed that older adults were less fatigable than young adults during an isometric intermittent fatiguing task of the knee extensors. This greater fatigue resistance was not maintained in very old adults independent of sex. Fatigability at exhaustion was likely due to impairments in contractile function for the three age groups. Keywords: Very old; Fatigue; Magnetic stimulation; Contractile function; Knee extensors. Y., Inoue, S., Matsudo, S.M., Mutrie, N., Oppert, J., Rowe, D.A., Schmidt, M.D., Schofield, G.M., Spence, J.C., Teixeira, P.J., Tully, M.A., and Blair, S.N., 2011. How

Summary of the study 2

In study 2 we observed similar total work (absolute performance) between young and old adults, with very old adults performing less total work. Young adults were stronger than the other two age groups, so for each contraction they performed more work. Older adults showed reduced performance fatigability at exhaustion and performed more contractions than young adults, which resulted in a similar absolute performance between those two age groups. Very old adults performed similar number of contraction than young adults but possessed lower strength, which resulted in lower total work compared to the other two age groups. Thus, this greater "fatigue resistance" observed in old adults was not maintained at very old age. This may be due to the important deteriorations at the skeletal muscle level, as very old adults presented similar impairments in contractile function than the others age groups despite lower total work produced, while no impairments in voluntary activation were observed. We did not find sex-related differences in performance or performance fatigability with age.

As we showed in study 1, activities such as walking could impact neuromuscular function with aging.

Walking requires the dynamic contractions of multiple muscular groups to sustain a load relative to the individuals' body weight. Thus, the results observed in study 2 might not be directly transferable to the activities of daily living. Indeed, when concentric tasks were used, performance fatigability has been shown to decrease with advancing age [e.g. (Sundberg et al. 2018b)]. When comparing young and old adults during a cycling task performed at similar total work, young adults presented lower performance fatigability, impairment in contractile function and no alterations in voluntary activation compared to the old adults (Weavil et al. 2018).

In study 3, we aimed to investigate performance and performance fatigability across age during isometric, concentric and cycling fatiguing tasks. To compare age groups in a more ecologically valid condition than in study 2, we set the workload as percentage of body weight.

Abstract (250 words)

The aim of the present study was to investigate performance and fatigability during isometric, concentric and cycling fatiguing tasks in young, old and very old men. Fifteen young, 13 old and 12 very old males randomly performed three fatiguing tasks: an isometric (ISO) and a concentric (CON) incremental fatigue tests on an isokinetic dynamometer and a similar test on a validated cycle ergometer (BIKE). ISO and CON consisted of stages of 75 contractions (0.8-s on/0.8-s off), and similar duration and duty cycle for BIKE (120-s at 37.5 rpm). Increments were device-specific and set as percentage of body weight. Percutaneous electrical nerve stimulation was used to evaluate voluntary activation (VA; i.e. interpolated twitch technique) and resting twitch amplitude (Tw) .

Performance (number of stages) was greater for young than old and very old adults for the three sessions, and greater for old than very old adults for BIKE only (P<0.001). The decrease in maximal force (i.e. performance fatigability) was greater for young than very old adults after ISO (P<0.001), but similar across age-groups after CON and BIKE. Tw amplitude decreased in the three sessions independent of age (P<0.001). VA decreased only for young adults in BIKE (P<0.05). These results showed that performance decreased with advancing age independent of the task when the load is set relative to the individual body weight. Performance fatigability was greater for young than very old adults during the isometric task, but similar between age groups during the dynamic tasks.

Performance fatigability was mainly driven by impairments in contractile function.

after the isometric fatiguing task only, and greater fatigability with advancing age after the concentric and cycling fatiguing tasks.

Methods

Participants

Fifteen young, 13 old and 12 very old men participated in the study and were clearly informed on the experimental procedures prior giving their written consent. Participants were free from psychological, musculoskeletal and neurological disorders or disability. Participants were asked to avoid exercise and alcohol consumption for 24h before each visit. Very old adults were needed to score more than 20 at the mini-mental state examination [mean score = 28 ± 3;(Jacqmin-Gadda et al. 1997)]. All the procedures were approved by the Comité de Protection des Personnes and were performed in accordance with the declaration of Helsinki (ClinicalTrials.gov, Identifier: NCT04516538). The university hospital of Saint-Etienne was the sponsor of the present study.

Experimental protocol

Participants visited the laboratory on four occasions separated by 7 days. During the first visit, all participants were familiarized with the procedures used to assess neuromuscular function and fatigability, and an accelerometer (wGT3X-BT; ActiGraph, Pensacola, USA) was given to wear for 7 consecutive days to objectively assess physical activity (Varesco et al. 2021b). The old and very old adults also performed a 6-minutes-walk-test (6MWT), which consisted in covering the longest distance possible in 6 minutes on a corridor of 30 meters (Crapo et al. 2002). Following the 6MWT, old and very old men performed a Timed-Up-and-Go (TUG) test, in which participants were required to stand up from a chair, walk a distance of three meters, turn around, return, and sit down on the chair (Mathias et al. 1986). The stopwatch was started when the back of the participants moved from the chair and stopped when the participants were seated on with their back on the back of the chair.

All data were analyzed offline using Labchart software (v8, ADInstruments). Performance was quantified as total number of stages performed until exhaustion. In case of incomplete stage at exhaustion, the proportion of the last stage was also considered (e.g. 7.5 stages). MVIC was defined as the highest 0.5-s window of the contraction. MVIC prior the QIF tests was also normalized to body weight. MVCC was defined as the maximal value recorded for each MVCC. Performance fatigability was defined as the percentage of reduction in MVIC and MVCC (when applicable). Voluntary activation (VA) was calculated using the following equation:

where SIT is the amplitude of the superimposed twitch evoked by peripheral nerve stimulation during MVIC, and Tw is the amplitude of the twitch evoked at rest. Changes in Tw were used to identify impairments in muscle contractile function. To evaluate neuromuscular propagation, changes in Mwave amplitude (Mmax) from baseline for the VL, RF and VM were analyzed from the EMG response to Tw.

Statistical Analysis

All variables are reported as mean ± SD in the text, tables and figures. Normality of data was checked using the Shapiro-Wilk test. Because measures from the cycle-ergometer showed a systematic ~30% difference compared to classic dynamometers (Doyle- Baker et al. 2017), data at baseline were analyzed separately for each session. Unpaired t-test were used to analyze differences between old and very old adults in the performance at the 6MWT and TUG test. One-way ANOVAs were used to analyze age-related differences in participants' characteristics (age, weight, height, physical activity, workload increments as %MVIC and %MVCC), neuromuscular data at baseline (absolute and normalized MVIC, MVCC, Tw, VA), %MVCC loss and performance. HR at exhaustion was analyzed using a mixed-effect ANOVA with session as within-group factor and age as between-

Summary of the study 3

In study 3, we observed a better performance for young adults at the three fatiguing tasks compared to old and very old adults. Old adults performed better than very old adults in the cycling task only.

Performance fatigability was similar at exhaustion for CON, whereas very old adults had a lower decrease in maximal force than young adults after ISO. Performance fatigability was similar across groups for the cycling task at exhaustion, but force loss of old and very old adults was greater than young adults at the early stages.

Performance fatigability was mainly driven by impairments in contractile function for the three conditions independent of age. In active older adults, voluntary activation might be preserved by physical activity and don't play a key role in the age-related differences in performance and performance fatigability at the QIF tests.

Because we showed that the age-related differences in performance and performance fatigability vary when the intensity is set relative to maximal force production capacity or body weight, future studies should consider studying the neuromuscular alterations with aging in more ecologically valid conditions.

In study 2, central and peripheral alterations were investigated using magnetic stimulations. We did not observe impairments in voluntary activation, while contractile function was similarly impaired in all three age groups. The extend of those impairments could vary across different fatiguing protocols adopted (Thomas et al. 2018). Similar to what was observed in the present study, previous investigations found impairments in contractile function, with limited impairments in voluntary activation (Callahan et al. 2009;Solianik et al. 2017). We confirmed those results during an incremental intermittent fatiguing task and extended those findings to very old adults. The ischemia caused by isometric contractions impedes oxygenated blood to reach the capillaries (Moritani et al. 1992), promoting impairments in contractile function (Copithorne and Rice 2020). The main impairments are due to mechanisms such as reduction in excitation-contraction coupling linked to high rates of ATP turnover, and accumulation of free phosphate and H + (Sundberg and Fitts 2019).

Because in very old adults the percentage of type I fibers was not related to performance or performance fatigability, similar loss in type I and type II muscle fibers, metabolites accumulation in the working fibers, and the recruitment of fast-twitch motor units could impair performance in this age group (Sundberg et al. 2019).

In study 3, participants performed a concentric and a cycling fatiguing task in addition to the isometric task. During and after the concentric task, similar performance fatigability was found across groups, while performance was greater for young than old and very old adults. The earlier impairments in contractile function during the task for old and very old adults compared to young adults suggest greater metabolite accumulation (Pi, H + and dihydrogen phosphate) within the working muscles of old adults (Sundberg et al. 2019), which may be induced by lower contraction efficiency in concentric mode with advancing age (Layec et al. 2018) and the greater workload relative to their MVCC. This rapid rise in contractile function impairment would have led to quickly reach task failure. The variability in performance fatigability could have masked some of the age-related impairments in maximal torque loss (Hunter et al. 2016)