Ana J Pinto
email: ana.pinto@cuanschutz.edu

Audrey Bergouignan
email: audrey.bergouignan@cuanschutz.edu

Paddy C Dempsey
email: paddy.dempsey@mrc-epid.cam.ac.uk

Hamilton Roschel

Neville Owen
email: neville.owen@baker.edu.au

Bruno Gualano
email: gualano@usp.br

David W Dunstan
email: david.dunstan@baker.edu.au

The Physiology of Sedentary Behavior

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6) Reducing/interrupting SB improves body composition, intermediary metabolism, and cardiovascular health outcomes, but effects are small, albeit marginally clinically meaningful. Most studies have been conducted in healthy population groups (i.e., outcomes within normal ranges) and larger effects may be observed in unhealthy populations.

7) The 'sit less, move more and exercise' focus of contemporary public health guidelines is a consensus based primarily on epidemiological findings, and further experimental evidence is needed to elucidate the physiological effects of interventions combining exercise and reduction/interruptions to SB. Nevertheless, reducing/interrupting SB is a low-risk strategy of clinical and population health relevance, and can serve as a stepping stone to regular participation in moderate-to-vigorous intensity PA.

INTRODUCTION

Sedentary behavior (SB) is defined as any waking behavior characterized by a low energy expenditure (≤1.5 metabolic equivalent of task [MET]) while sitting or lying down [START_REF] Tremblay | SBRN Terminology Consensus Project Participants. Sedentary Behavior Research Network (SBRN) -Terminology Consensus Project Process and Outcome[END_REF]. Driven by environmental, economic, social, and technological changes, SB is now understood to be a major component of the human movement spectrum that can impact health adversely [START_REF] Bull | World Health Organization 2020 Guidelines on Physical Activity and Sedentary Behaviour[END_REF][START_REF] Katzmarzyk | Sedentary Behavior and Health: Update from the 2018 Physical Activity Guidelines Advisory Committee[END_REF]. In adults and older adults, time spent sedentary can range from 5 to 11.5 h/day [START_REF] Ekelund | Dose-Response Associations between Accelerometry Measured Physical Activity and Sedentary Time and All Cause Mortality: Systematic Review and Harmonised Meta-Analysis[END_REF][START_REF] Bauman | The Descriptive Epidemiology of Sitting. A 20-Country Comparison Using the International Physical Activity Questionnaire (IPAQ)[END_REF][START_REF] Bennie | The Prevalence and Correlates of Sitting in European Adults -a Comparison of 32 Eurobarometer-Participating Countries[END_REF][START_REF] De | Original Research Socio-Demographic Patterning of Self-Reported Physical Activity and Sitting Time in Latin American Countries: Findings from ELANS[END_REF].

Particular attention has been given to SB only since the early 2000's when the term "inactivity physiology" (i.e., acute and chronic physiological effects of SB [nonexercise activity deficiency]) was put forward by Hamilton and colleagues as a separate research field from exercise physiology [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF][START_REF] Hamilton | Too Little Exercise and Too Much Sitting: Inactivity Physiology and the Need for New Recommendations on Sedentary Behavior[END_REF]. This shift was motivated by experimental findings demonstrating key differences in mechanisms driving skeletal muscle lipoprotein lipase (LPL) responses between physical inactivity and exercise compared to normal standing and ambulatory activity in rats [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF]. The rapid accumulation of experimental evidence on SB over the past 20 years has built upon these early insights, with greater attention being directed at understanding the health consequences of daily hours spent sedentary and the countermeasure strategies aimed at reducing/interrupting time spent sedentary.

This review provides a perspective on: (i) how to characterize SB; (ii) the pros and cons of the currently available experimental models employed in the investigation of physiology of SB; (iii) the physiological effects of variations in SB and potential underlying mechanisms; and, (iv) the gaps that currently exist in the scientific understanding of the physiology of SB. For a broad and practically-informed perspective, we address the extent to which the physiological evidence base can help to further focus and sharpen public health and clinical practice guidelines, extending beyond the well-understood and accepted 'exercise more' message, towards a more comprehensive 'sit less, move more and exercise' message.

AN OPERATIONAL FRAMEWORK FOR SEDENTARY BEHAVIOR PHYSIOLOGY

RESEARCH

The term SB, derived from the Latin word sedere ('to sit'), refers to any waking behavior posture [START_REF] Tremblay | SBRN Terminology Consensus Project Participants. Sedentary Behavior Research Network (SBRN) -Terminology Consensus Project Process and Outcome[END_REF] (see TABLE 1 for key terms in SB research). First, we discuss key features and themes of SB research. This operational framework provides guidance for more indepth considerations on the physiology of SB later in the review.

- -------------------------------INSERT TABLE 1 ABOUT HERE --------------------------------

Characteristics of sedentary behavior

SBs are identified based on their physiological and postural characteristics.

Physiologically, the average energy cost of common types of SB ranges between 1.0 and 1.5 METs in healthy adults [START_REF] Ainsworth | Compendium of Physical Activities: A Second Update of Codes and MET Values[END_REF] during fasting [START_REF] Judice | What Is the Metabolic and Energy Cost of Sitting, Standing and Sit/Stand Transitions?[END_REF][START_REF] Betts | The Energy Cost of Sitting Versus Standing Naturally in Man[END_REF][START_REF] Gao | Acute Metabolic Response, Energy Expenditure, and EMG Activity in Sitting and Standing[END_REF] and postprandial states [START_REF] Mansoubi | Energy Expenditure During Common Sitting and Standing Tasks: Examining the 1.5 MET Definition of Sedentary Behaviour[END_REF][START_REF] Newton | The Energy Expenditure of Sedentary Behavior: A Whole Room Calorimeter Study[END_REF], as measured by indirect calorimetry [START_REF] Judice | What Is the Metabolic and Energy Cost of Sitting, Standing and Sit/Stand Transitions?[END_REF][START_REF] Betts | The Energy Cost of Sitting Versus Standing Naturally in Man[END_REF][START_REF] Gao | Acute Metabolic Response, Energy Expenditure, and EMG Activity in Sitting and Standing[END_REF][START_REF] Mansoubi | Energy Expenditure During Common Sitting and Standing Tasks: Examining the 1.5 MET Definition of Sedentary Behaviour[END_REF] or by whole-room calorimetry [START_REF] Newton | The Energy Expenditure of Sedentary Behavior: A Whole Room Calorimeter Study[END_REF].

Overall, energy expenditure during sitting is higher than reclining [START_REF] Betts | The Energy Cost of Sitting Versus Standing Naturally in Man[END_REF][START_REF] Newton | The Energy Expenditure of Sedentary Behavior: A Whole Room Calorimeter Study[END_REF], but lower than standing [START_REF] Judice | What Is the Metabolic and Energy Cost of Sitting, Standing and Sit/Stand Transitions?[END_REF][START_REF] Betts | The Energy Cost of Sitting Versus Standing Naturally in Man[END_REF] and lower than all intensities of physical activity (PA) [START_REF] Mansoubi | Energy Expenditure During Common Sitting and Standing Tasks: Examining the 1.5 MET Definition of Sedentary Behaviour[END_REF]. For heart rate (HR), similar responses are observed [START_REF] Judice | What Is the Metabolic and Energy Cost of Sitting, Standing and Sit/Stand Transitions?[END_REF][START_REF] Gao | Acute Metabolic Response, Energy Expenditure, and EMG Activity in Sitting and Standing[END_REF].

In skeletal muscle, increased contractile activity is required to sustain standing and ambulatory activities [START_REF] Pesola | Muscle Inactivity Is Adversely Associated with Biomarkers in Physically Active Adults[END_REF]; consequently, blood flow increases and the metabolic demands of the contracting muscles are accommodated [START_REF] Joyner | Regulation of Increased Blood Flow (Hyperemia) to Muscles During Exercise: A Hierarchy of Competing Physiological Needs[END_REF]. In contrast, muscle contractile activity during sitting postures (as measured by electromyographic [EMG] activity) is significantly lower than for standing and ambulatory activities [START_REF] Gao | Acute Metabolic Response, Energy Expenditure, and EMG Activity in Sitting and Standing[END_REF][START_REF] Pesola | Muscle Inactivity Is Adversely Associated with Biomarkers in Physically Active Adults[END_REF][START_REF] Tikkanen | Muscle Activity and Inactivity Periods During Normal Daily Life[END_REF].

EMG activity in the quadriceps and hamstring muscle groups is ~2.0-2.5, ~7.0-10.5 and ~18.0 times higher during standing, walking and stair climbing in daily living settings, respectively, as compared to sitting [START_REF] Pesola | Muscle Inactivity Is Adversely Associated with Biomarkers in Physically Active Adults[END_REF][START_REF] Tikkanen | Muscle Activity and Inactivity Periods During Normal Daily Life[END_REF]. To match the reduced metabolic demands of low muscle activity, skeletal muscle blood flow is also significantly lower and less variable during sitting as compared to standing and ambulatory activities [START_REF] Joyner | Regulation of Increased Blood Flow (Hyperemia) to Muscles During Exercise: A Hierarchy of Competing Physiological Needs[END_REF][START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF][START_REF] Peddie | The Effects of Prolonged Sitting, Prolonged Standing, and Activity Breaks on Vascular Function, and Postprandial Glucose and Insulin Responses: A Randomised Crossover Trial[END_REF].

Elements pertaining to SB that may explain the differential impacts of SB on health outcomes include frequency, intensity, time, and type, the so-called FITT principle (TABLE 2) [START_REF] Tremblay | Physiological and Health Implications of a Sedentary Lifestyle[END_REF]. Engagement in SB can be accordingly described and monitored, which aligns with and builds on the FITT principle for exercise prescription.

These key postural, physiological, and behavioral features are what define/characterize SB and should be considered explicitly in SB research.

--------------------------------INSERT TABLE 2 ABOUT HERE --------------------------------

Sedentary behavior versus physical inactivity

The term 'sedentary' had previously been used interchangeably with physical inactivity to denote insufficient levels of moderate-to-vigorous intensity PA (MVPA), i.e., not achieving the current PA guidelines [START_REF] Tremblay | SBRN Terminology Consensus Project Participants. Sedentary Behavior Research Network (SBRN) -Terminology Consensus Project Process and Outcome[END_REF]. However, SB and physical inactivity are now viewed as being separate entities on a continuum of human movement and nonmovement behaviors [START_REF] Tremblay | SBRN Terminology Consensus Project Participants. Sedentary Behavior Research Network (SBRN) -Terminology Consensus Project Process and Outcome[END_REF]. According to this definition, a person could be classified as being both highly sedentary and physically active. This points to four distinct classifications: being physically active and highly sedentary, physically active and slightly sedentary, physically inactive and highly sedentary, or physically inactive and slightly sedentary (FIGURE 1). The importance of addressing the behavioral phenotype of both excessive SB and physical inactivity is now embodied in contemporary public health PA guidelines [START_REF] Bull | World Health Organization 2020 Guidelines on Physical Activity and Sedentary Behaviour[END_REF][START_REF] Piercy | The Physical Activity Guidelines for Americans[END_REF].

- ---------------------------------INSERT FIGURE 1 ABOUT HERE -----------------------------------From a physiological perspective, differentiating between 'SB' and 'physical inactivity' may initially seem rather semantic. Indeed, reviews have already summarized the evidence to date on numerous physiological responses of increasing SB [START_REF] Dempsey | Sitting Less and Moving More: Implications for Hypertension[END_REF][START_REF] Dempsey | Sitting Less and Moving More: Improved Glycaemic Control for Type 2 Diabetes Prevention and Management[END_REF][START_REF] Dunstan | Sit Less and Move More for Cardiovascular Health: Emerging Insights and Opportunities[END_REF][START_REF] Homer | Too Much Sitting and Dysglycemia: Mechanistic Links and Implications for Obesity[END_REF][START_REF] Wheeler | Sedentary Behavior as a Risk Factor for Cognitive Decline? A Focus on the Influence of Glycemic Control in Brain Health[END_REF][START_REF] Padilla | Prolonged Sitting Leg Vasculopathy: Contributing Factors and Clinical Implications[END_REF] and it is evident that many of these relate to the responses following imposed physical inactivity [START_REF] Bergouignan | Physical Inactivity as the Culprit of Metabolic Inflexibility: Evidences from Bed-Rest Studies[END_REF][START_REF] Thyfault | Metabolic Disruptions Induced by Reduced Ambulatory Activity in Free-Living Humans[END_REF][START_REF] Booth | Role of Inactivity in Chronic Diseases: Evolutionary Insight and Pathophysiological Mechanisms[END_REF][START_REF] Booth | Lack of Exercise Is a Major Cause of Chronic Diseases[END_REF]. Even though the effects of SB and physical inactivity are generally in a similar direction (deleterious), excessive participation in SB has been shown to result in adverse effects even in the presence of large volumes of aerobic and/or resistance exercise (a sedentary yet physically active condition) [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]. Exercising (above current PA guidelines) during bed rest does not necessarily counteract, or only partially counteracts, some of the adverse effects of large volumes of SB in healthy adults [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF].

These findings point not only to likely independent adverse health effects of SB, but also to the potential benefits of regular non-exercise activity and/or muscle contractions.

Sedentary behavior physiology research: The seminal role of 'inactivity physiology' and animal studies

The term 'inactivity physiology' was first proposed in the early 2000's as a separate field and distinct from exercise physiology [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Too Little Exercise and Too Much Sitting: Inactivity Physiology and the Need for New Recommendations on Sedentary Behavior[END_REF][START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF]. The premise was that excessive SB was not the same as lack of exercise and that SBs have their own unique physiological consequences.

Research in the 'inactivity physiology' context has been examined primarily using hindlimb unloading and wheel lock methodologies in rats. The key objective of these studies was to better understand how increasing SB and imposing physical inactivity (or rather hypodynamia and hypokinesia) may trigger maladaptations linked to chronic diseases. Here, key findings pertinent to SB physiology are summarized.

Hindlimb unloading models involve suspending rodents by their tail, thereby preventing any weight-bearing activities of the lower limbs. Hindlimb unloading studies have reported rapid development of insulin resistance (increased glucose-insulin index) after one day of limb suspension [START_REF] O'keefe | Development of Whole-Body and Skeletal Muscle Insulin Resistance after One Day of Hindlimb Suspension[END_REF]. Notably, glucose transport activity and intramuscular triglycerides were significantly lower in soleus muscle, but not in extensor digitorum longus after one day of limb suspension, which was driven by increases in p38 mitogen-activated protein kinase expression (MAPK), known to negatively interact with insulin signaling cascade [START_REF] O'keefe | Development of Whole-Body and Skeletal Muscle Insulin Resistance after One Day of Hindlimb Suspension[END_REF]. This suggests that those muscles which predominantly contain type 1 fibers are more susceptible to maladaptations related to increasing SB than muscles composed of type 2 fibers predominantly. This has also been shown in humans following periods of bed rest. For example, plantar flexor and monoarticular knee extensor muscles were found to be more affected than hip extensor/adductor muscles after head-down bed rest (HDBR) and horizontal bed rest [START_REF] Belavy | Differential Atrophy of the Lower-Limb Musculature During Prolonged Bed-Rest[END_REF][START_REF] Berg | Hip, Thigh and Calf Muscle Atrophy and Bone Loss after 5-Week Bedrest Inactivity[END_REF][START_REF] Mendis | Effect of Prolonged Bed Rest on the Anterior Hip Muscles[END_REF][START_REF] Miokovic | Differential Atrophy of the Postero-Lateral Hip Musculature During Prolonged Bedrest and the Influence of Exercise Countermeasures[END_REF]. In contrast, the biarticular knee extensor and hip flexor rectus femoris, other anteromedial hip muscles and short head of biceps femoris were found to be comparatively less affected by horizontal bed rest [START_REF] Belavy | Differential Atrophy of the Lower-Limb Musculature During Prolonged Bed-Rest[END_REF]. Notably, faster rates of muscle atrophy were observed in anti-gravity muscles and those that are more intensively required for standing and walking [START_REF] Belavy | Differential Atrophy of the Lower-Limb Musculature During Prolonged Bed-Rest[END_REF]. Similarly, myosin heavy chain (MHC) distribution in the skeletal muscle shifted from slow-twitch (MHC I) towards hybrid (I/IIa and IIa/IIx) and fast-twitch (IIa and IIx) fiber types in vastus lateralis following 35-84 days of HDBR and horizontal bed rest [START_REF] Borina | Myosin and Actin Content of Human Skeletal Muscle Fibers Following 35 Days Bed Rest[END_REF][START_REF] Gallagher | Effects of 84-Days of Bedrest and Resistance Training on Single Muscle Fibre Myosin Heavy Chain Distribution in Human Vastus Lateralis and Soleus Muscles[END_REF][START_REF] Trappe | Human Single Muscle Fibre Function with 84 Day Bed-Rest and Resistance Exercise[END_REF]. Similar alterations in slow and hybrid fibers, but not fast fibers, were observed in soleus muscle after 84 days of HDBR [START_REF] Gallagher | Effects of 84-Days of Bedrest and Resistance Training on Single Muscle Fibre Myosin Heavy Chain Distribution in Human Vastus Lateralis and Soleus Muscles[END_REF].

Hamilton and colleagues showed that distinctive physiological pathways are activated with hindlimb unloading, particularly LPL activity, which seems to remain largely unaffected by MVPA [START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF]. Rat skeletal muscle triglyceride uptake was reduced by 75% and LPL protein expression and enzymatic activity were rapidly suppressed during acute (1-18 h) and chronic (~10 h/day over 11 days) periods of hindlimb unloading [START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF]. Alterations in heparin-released and intracellular LPL activity decreased mono-exponentially in both the soleus (type 1) and red quadriceps (predominantly type 2) muscles after 12 hours of limb unloading. These alterations were rapidly reversed with light-intensity contractile activity in both soleus and quadriceps muscles [START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF][START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF][START_REF] Bey | Patterns of Global Gene Expression in Rat Skeletal Muscle During Unloading and Low-Intensity Ambulatory Activity[END_REF].

Interestingly, MVPA/exercise training did not enhance LPL regulation in type 1 muscles and type 2 muscles that were not recruited during running [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Local Contractile Activity and Muscle Fiber Type on LPL Regulation During Exercise[END_REF]. In type 1 muscles recruited during running, there was an increase in heparin-released LPL activity, LPL mRNA level, and LPL protein mass [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Local Contractile Activity and Muscle Fiber Type on LPL Regulation During Exercise[END_REF]. Additionally, heparin-released LPL activity was ten-fold less in the soleus and quadriceps muscles and two-fold less in the rectus femoris muscle of rats subjected to 12-hour limb unloading as compared with lowintensity ambulatory controls [START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF]. The absence of changes in LPL activity in the heart and diaphragm, both muscles with high oxidative capacity, also suggested loss of muscle LPL activity was constrained to unused muscles [START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF].

Despite changes in LPL activity, no changes were observed in skeletal muscle LPL gene expression following acute (1-18 h) and sustained (~10 h/day over 11 days) periods of hindlimb unloading [START_REF] Bey | Patterns of Global Gene Expression in Rat Skeletal Muscle During Unloading and Low-Intensity Ambulatory Activity[END_REF]. However, Zderic and Hamilton (47) demonstrated that skeletal muscle differentially expresses at least 17 genes involved in homeostasis in humans and rats. Of particular interest, Lipid Phosphatase-1 (LPP1/PAP2A), a key gene for degradation of prothrombotic and proinflammatory lysophospholipids, was suppressed locally in muscle tissue after 12 hours of hindlimb unloading in rats, and after 12 hours of prolonged sitting in humans [START_REF] Zderic | Identification of Hemostatic Genes Expressed in Human and Rat Leg Muscles and a Novel Gene (LPP1/PAP2A) Suppressed During Prolonged Physical Inactivity (Sitting)[END_REF]. Of note, exercise was ineffective at counteracting this decrease in both species [START_REF] Zderic | Identification of Hemostatic Genes Expressed in Human and Rat Leg Muscles and a Novel Gene (LPP1/PAP2A) Suppressed During Prolonged Physical Inactivity (Sitting)[END_REF].

Wheel lock models involve periods of habitual or voluntary activity (3-6 weeks; typically, 5-10 km/day of running) which is suddenly restricted (i.e., running wheel locked) to permit only minimal movement within the cage for up to 7 days. While daily wheel running increased insulin-dependent glucose uptake in isolated skeletal muscle, a rapid decrease in insulin sensitivity was reported following only 2 days of wheel lock in rats [START_REF] Kump | Alterations in Insulin Receptor Signalling in the Rat Epitrochlearis Muscle Upon Cessation of Voluntary Exercise[END_REF]. This reduction in insulin-dependent glucose transport was associated with reduced activation of the insulin-signaling pathway and glucose transporter 4 (GLUT-4) protein content. Pronounced gains in intra-abdominal fat mass (25 to 48%) were also reported following 1 week of wheel lock [START_REF] Laye | Inactivity Induces Increases in Abdominal Fat[END_REF][START_REF] Kump | Sustained Rise in Triacylglycerol Synthesis and Increased Epididymal Fat Mass When Rats Cease Voluntary Wheel Running[END_REF]. Interestingly, lowering food intake during wheel lock did not significantly change fat mass increase compared to the rats that were fed ad libitum, indicating that fat storage was the result of SB and physical inactivity per se, rather than positive energy balance [START_REF] Laye | Inactivity Induces Increases in Abdominal Fat[END_REF]. Despite providing important initial insights for SB research, wheel lock models are considered to be extreme models of inactivity in which animals transit from very high daily amounts of exercise to sedentariness/inactivity. Therefore, it may be more a model of detraining from exercise rather than a model to study adaptations to increasing SB.

Evidence from 'inactivity physiology' studies using hindlimb unloading, and wheel lock methodologies have been instrumental in laying the foundation for experimental studies related to SB and physical inactivity physiology in humans by providing initial evidence on the potential adverse effects and underpinning mechanisms associated with these behaviors as compared to habitual activity and exercise.

Role of sedentary behavior in health and disease

Extensive epidemiological evidence has highlighted that excessive daily time in SB is associated with increased risk of early mortality and chronic diseases, including obesity, type 2 diabetes, cardiovascular disease, metabolic syndrome, certain type of cancers, and others [START_REF] Ekelund | Dose-Response Associations between Accelerometry Measured Physical Activity and Sedentary Time and All Cause Mortality: Systematic Review and Harmonised Meta-Analysis[END_REF][START_REF] Diaz | Patterns of Sedentary Behavior and Mortality in U.S. Middle-Aged and Older Adults: A National Cohort Study[END_REF][START_REF] Nicholas | Leisure-Time Physical Activity Does Not Attenuate the Association between Occupational Sedentary Behavior and Obesity: Results from Alberta's Tomorrow Project[END_REF][START_REF] Van Der Berg | Associations of Total Amount and Patterns of Sedentary Behaviour with Type 2 Diabetes and the Metabolic Syndrome: The Maastricht Study[END_REF][START_REF] Wilmot | Sedentary Time in Adults and the Association with Diabetes, Cardiovascular Disease and Death: Systematic Review and Meta-Analysis[END_REF][START_REF] Patterson | Sedentary Behaviour and Risk of All-Cause, Cardiovascular and Cancer Mortality, and Incident Type 2 Diabetes: A Systematic Review and Dose Response Meta-Analysis[END_REF][START_REF] Engelen | Who Is at Risk of Chronic Disease? Associations between Risk Profiles of Physical Activity, Sitting and Cardio-Metabolic Disease in Australian Adults[END_REF][START_REF] Lynch | Sedentary Behavior and Cancer: A Systematic Review of the Literature and Proposed Biological Mechanisms[END_REF][START_REF] Matthews | Amount of Time Spent in Sedentary Behaviors and Cause-Specific Mortality in US Adults[END_REF][START_REF] Falck | What Is the Association between Sedentary Behaviour and Cognitive Function? A Systematic Review[END_REF]. Yet mechanisms involved in this increased risk are poorly understood.

A systematic review synthesized current knowledge of the associations of SB with gene expression and epigenetic modifications in children and adolescents. Overall, evidence is still limited, but some studies suggest candidate genes and non-coding ribonucleic acids (RNAs) that are linked to/regulated by SB, including higher miRNA-222 and miRNA-146a levels (related to angiogenesis and inflammation), and methylation at HSD11B2 promoter (related to stress/cortisol metabolism) [START_REF] Plaza-Florido | Transcriptional and Epigenetic Response to Sedentary Behavior and Physical Activity in Children and Adolescents: A Systematic Review[END_REF].

Additionally, screen time was a significant moderator in the association of the rs9939609 single nucleotide polymorphism (SNP) located on the fat mass and obesityassociated gene (FTO) with metabolic syndrome clustered cardiometabolic risk score in children and adolescents of low cardiorespiratory fitness [START_REF] Sehn | What Is the Role of Cardiorespiratory Fitness and Sedentary Behavior in Relationship between the Genetic Predisposition to Obesity and Cardiometabolic Risk Score?[END_REF]. In adults, FTO SNP rs9939609 was significantly associated with self-reported time spent in SB, and sedentary time partially mediated the association between FTO and body mass index (BMI) [START_REF] Klimentidis | FTO Association and Interaction with Time Spent Sitting[END_REF]. In contrast, another study demonstrated that the association between objectively measured SB and FTO SNP rs17817449 was fully attenuated by BMI in adults, suggesting the association between SB and FTO was explained by adiposity [START_REF] Martorell | The FTO RS17817449 Polymorphism Is Not Associated with Sedentary Time, Physical Activity, or Cardiorespiratory Fitness: Findings from the Genadio Cross-Sectional Study[END_REF]. Although the mechanisms through which FTO increases BMI and adiposity have not been elucidated, knockout mice models suggest that FTO may be involved in energy homeostasis via regulation of energy expenditure [START_REF] Fischer | Inactivation of the FTO Gene Protects from Obesity[END_REF].

Ascribing causality from observational evidence is difficult. To overcome this limitation some studies have used Mendelian randomization, which is a well-established tool that employs genetic variants as instrumental variables for exposures (e.g., SB and PA). Since the genetic variants are randomly assigned during meiosis, Mendelian randomization can minimize confounding and reverse causation, potentially providing stronger evidence for causal inference [START_REF] Smith | Mendelian Randomization: Genetic Anchors for Causal Inference in Epidemiological Studies[END_REF]. Overall, findings from Mendelian randomization studies are aligned with those from observational studies. Totals of 136, 43 and 5 genetic SNPs have been found to be associated with leisure-time TV watching, computer use, and driving, respectively. Genetically predicted duration of TV watching was positively associated with risk of myocardial infarction, heart failure and atrial fibrillation, which remained significant after adjustments for genetically predicted PA. Associations between computer time use and driving and cardiovascular diseases were inconsistent and non-significant [START_REF] Yang | Genetic Liability to Sedentary Behavior in Relation to Myocardial Infarction and Heart Failure: A Mendelian Randomization Study[END_REF]. Similarly, another study identified a total of 89 genetic SNPs that were associated with TV watching.

Genetically predicted duration of TV watching, but not computer use and driving, was positively associated with risk of developing type 2 diabetes [START_REF] Deng | Physical Activity, Sedentary Behavior, and the Risk of Type 2 Diabetes: A Two-Sample Mendelian Randomization Analysis in the European Population[END_REF]. A study using individual-level data from 130,957 females identified 6 SNPs as predictors of participation in SB. Females with genetic variants predisposing them to a higher time in SB had a higher risk of hormone-receptor-negative and in situ breast cancer. Subanalysis suggested that SB and PA independently influence risk of breast cancer [START_REF] Dixon-Suen | Activity and Sedentary Behavior Recommendations[END_REF].

Finally, a genome-wide association study (GWAS) of PA and SB provided some insights into underlying mechanism and roles in disease prevention (69). Eighty-eight loci (89 independent SNPs) were associated with leisure screen time, 8 loci for SB at work, and none for SB during commuting. Authors observed significant genetic correlation between high leisure screen time and higher adiposity-related traits, particularly fat percentage, and poor cardiometabolic status, including higher triglycerides, cholesterol, fasting glucose and insulin concentrations, and odds of type 2 diabetes, coronary artery disease, cancer, worsened mental health outcomes, and decreased longevity. Additionally, Mendelian randomization has consistently shown that leisure screen time and BMI causally influence each other, with the causal role of leisure screen time in BMI being two-to-threefold larger than the effect of BMI on leisure screen time. In this same study, tissue and cell-type enrichment analysis has also suggested a role for visual information processing and the reward system in leisure screen time. Leisure screen time-associated loci were mildly enriched for genes whose expression in skeletal muscle is altered by resistance exercise training. Forty-six candidate genes pointed to pathways related to endocytosis, locomotion, and myopathy, but in vivo models are required to confirm or refuse a role in SB. Overall, such causal inferences can support public health message that increasing MVPA and reducing SB mitigate risk of multiple chronic diseases (69).

The summary of available literature highlights the small evidence base with respect to the mechanisms behind the relationship between SB, health, and disease.

Very few genes and genetic regions have been studied in SB research. Despite the limited evidence, Mendelian randomization studies provide genetic support for a causal relationship between SB and risk of chronic diseases. There is a need for larger cohort studies and randomized controlled trials using 'omics' approaches (e.g., genomics, epigenomics, transcriptomics, proteomics, and metabolomics) to better understand the molecular mechanisms underlying the effects of SB on heath and disease.

The influence of sedentary behaviors on the relationship between exercise and health outcomes

Recent experimental evidence indicates excessive sedentary time also affects the relationship between participation in MVPA/exercise and health benefits (70)[START_REF] Ekelund | Joint Associations of Accelerometer Measured Physical Activity and Sedentary Time with All-Cause Mortality: A Harmonised Meta-Analysis in More Than 44 000 Middle-Aged and Older Individuals[END_REF][START_REF] Kim | Prolonged Sitting Negatively Affects the Postprandial Plasma Triglyceride-Lowering Effect of Acute Exercise[END_REF][START_REF] Akins | Inactivity Induces Resistance to the Metabolic Benefits Following Acute Exercise[END_REF][START_REF] Burton | Daily Step Count and Postprandial Fat Metabolism[END_REF].

Coyle and colleagues have demonstrated adverse effects of excessive participation in SB on postprandial metabolic responses and metabolic benefits of acute exercise [START_REF] Kim | Prolonged Sitting Negatively Affects the Postprandial Plasma Triglyceride-Lowering Effect of Acute Exercise[END_REF][START_REF] Akins | Inactivity Induces Resistance to the Metabolic Benefits Following Acute Exercise[END_REF][START_REF] Burton | Daily Step Count and Postprandial Fat Metabolism[END_REF]. Two or four days of prolonged sitting (>14 h/day and ~1,650 steps) resulted in increased postprandial plasma triglyceride responses regardless of energy intake, compared to 4 days of standing/walking (~8.4 h/day of SB and ~17,000 steps/day) in healthy, physically active males. Importantly, this altered response was not attenuated by a subsequent acute 1-hour bout of MVPA (~67% VO 2max ) performed at 1700h of day 4 [START_REF] Kim | Prolonged Sitting Negatively Affects the Postprandial Plasma Triglyceride-Lowering Effect of Acute Exercise[END_REF]. Using a similar study design, Akins and colleagues [START_REF] Akins | Inactivity Induces Resistance to the Metabolic Benefits Following Acute Exercise[END_REF] demonstrated that acute exposure to prolonged sitting (~13.5 h/day of sitting and <4,000 steps/day) not only prevented the traditional exercise-related benefits in postprandial triglycerides responses, but also improvements in postprandial plasma glucose and insulin responses in healthy adults. Furthermore, Burton & Coyle (74) compared postprandial plasma triglycerides responses to an acute exercise bout after 2 days of low (~2,500 steps/day), limited (~5,000 steps/day), or normal (~8,500 steps/day) daily step count to determine the range of step counts that elicited this blunted postprandial metabolic response to acute exercise. Following low and limited step counts, postprandial triglyceride responses were elevated by 22-23% and whole-body fat oxidation was reduced by 14-19% as compared to normal step count in healthy adults [START_REF] Burton | Daily Step Count and Postprandial Fat Metabolism[END_REF]. This finding indicates that altered metabolic responses to acute exercise can occur in those taking ~5,000 steps/day or lower.

These studies provide initial insights into a unique perspective that excessive SB might be a health hazard, not only via the physiological maladaptations that occur during sitting, but also by impacting the health benefits provided by MVPA/exercise.

Collectively, these findings highlight the need for: (i) addressing large amounts of time spent in SB to minimize/counteract its adverse effects; (ii) examining the physiological responses and adaptations within and across each of these distinct behavioral constructs, as there may be differential, additive and/or interacting physiological effects to consider.

These are research questions that arise from SB physiology that have not been pursued by exercise and physical inactivity physiology. A key feature in SB research has been to focus on shifting the balance of participation in SB towards LPA, rather than solely focusing on MVPA. This has also been included in the development of countermeasures to specifically address SB, with a growing body of experimental studies aiming to reduce and interrupt prolonged SB with various types of PA [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF][START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF][START_REF] Shrestha | Effectiveness of Interventions for Reducing Non-Occupational Sedentary Behaviour in Adults and Older Adults: A Systematic Review and Meta-Analysis[END_REF].

The understanding of the physiological impact of the interdependent relationships between SB, LPA and MVPA is a more recent focus and available evidence has been limited to the abovementioned studies. In the next subsection, the pros and cons of the relevant experimental models will be discussed in the context of their potential implications for investigations of the physiology of SB.

Experimental models with relevance for sedentary behavior research

SB-induced physiological changes in humans can be inferred from a variety of experimental modelsbed rest, limb immobilization/casting, reduced daily step count, reducing/interrupting prolonged sitting, and others (e.g., detraining, confinement, and natural experiments)that collectively induce variations in time spent in SB (FIGURE 2). Each of these approaches can provide complementary information related to the impacts of SB on health outcomes. Identifying and understanding the different goals, methodologies and assumptions that can be made under these models is fundamental when attempting to generalize their findings to SB physiology research.

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-------------------------------INSERT FIGURE 2 ABOUT HERE --------------------------------

Bed rest

Bed rest is a common practice within medical treatment for selected conditions. In research, bed rest was initially developed in the context of space exploration as a ground-based model used to mimic on Earth the physiological effects of microgravity.

The bed rest model is characterized by a postural change (lying down or lying down combined with head tilt [i.e., HDBR]) and lack of muscle contraction for extended periods of time. In the quest of mitigating the adverse health effects of microgravity on the body, space agencies have developed and tested countermeasures during bed rest studies including different exercise training protocols [START_REF] Bergouignan | Physical Inactivity as the Culprit of Metabolic Inflexibility: Evidences from Bed-Rest Studies[END_REF][START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF][START_REF] Rynders | Sedentary Behaviour Is a Key Determinant of Metabolic Inflexibility[END_REF][START_REF] Hodgson | Older Adults Are Not More Susceptible to Acute Muscle Atrophy after Immobilisation Compared to Younger Adults: A Systematic Review[END_REF][START_REF] Sonne | Endothelial Function after 10 Days of Bed Rest in Individuals at Risk for Type 2 Diabetes and Cardiovascular Disease[END_REF][START_REF] Sonne | Effect of 10 Days of Bedrest on Metabolic and Vascular Insulin Action: A Study in Individuals at Risk for Type 2 Diabetes[END_REF][START_REF] Friedrichsen | Muscle Inflammatory Signaling in Response to 9 Days of Physical Inactivity in Young Men with Low Compared with Normal Birth Weight[END_REF][START_REF] Mortensen | Physical Inactivity Affects Skeletal Muscle Insulin Signaling in a Birth Weight-Dependent Manner[END_REF].

Beyond the interest in space science, bed rest models have implications for SB and physical inactivity research. Bed rest has the advantage of taking place in a highly controlled environment, which allows accurate monitoring of the activity performed and of food intake. The degree of physical inactivity observed during bed rest may be seen as too extreme compared with that seen in the general population, who spend more time sitting with some level of body movement rather than strictly lying down. However, because of upper body movements and fidgeting movements in the bed, the PA level (i.e., the ratio between total and resting energy expenditures) measured during bed rest studies is similar to that of sedentary individuals (i.e., 1.4 to 1.5) [START_REF] Bergouignan | Regulation of Energy Balance During Long-Term Physical Inactivity Induced by Bed Rest with and without Exercise Training[END_REF][START_REF] Blanc | Energy and Water Metabolism, Body Composition, and Hormonal Changes Induced by 42 Days of Enforced Inactivity and Simulated Weightlessness[END_REF]. In contrast, strict bed rest involves prolonged exposures to the lying down position, which distinctly affects organs and physiological systems as compared to the free-living context, where sitting is the most predominant type of SB. For example, lower-limb muscles and weight-bearing muscles are more affected than upper limbs and non-weight-bearing muscles during bed rest [START_REF] Belavy | Differential Atrophy of the Lower-Limb Musculature During Prolonged Bed-Rest[END_REF][START_REF] Dirks | One Week of Bed Rest Leads to Substantial Muscle Atrophy and Induces Whole-Body Insulin Resistance in the Absence of Skeletal Muscle Lipid Accumulation[END_REF][START_REF] Leblanc | Regional Changes in Muscle Mass Following 17 Weeks of Bed Rest[END_REF]. Shift of fluid from the lower to upper part of the body is a unique adaptation to the lying down position, particularly in HDBR [START_REF] Pavy-Le Traon | From Space to Earth: Advances in Human Physiology from 20 Years of Bed Rest Studies (1986-2006)[END_REF], which may distinctly affect cardiovascular and central nervous systems which is in contrast to how SB manifests in the free-living context.

Immobilization

Limb immobilization/casting models are characterized by periods during which a limb is physically immobilized [START_REF] Adams | Skeletal Muscle Unweighting: Spaceflight and Ground-Based Models[END_REF][START_REF] Yasuda | Sex-Based Differences in Skeletal Muscle Function and Morphology with Short-Term Limb Immobilization[END_REF]. In the case of lower-limb immobilization, participants are commonly supported by crutches and asked to refrain from weight-bearing activity on the immobilized leg. Consequently, there is an extensive restriction of motion for the target limb with a reduction in habitual ambulatory activity, which ultimately results in local muscle disuse [START_REF] Adams | Skeletal Muscle Unweighting: Spaceflight and Ground-Based Models[END_REF]. The main focus of contemporary studies has been to investigate mechanisms underlying muscle disuse atrophy [START_REF] Yasuda | Sex-Based Differences in Skeletal Muscle Function and Morphology with Short-Term Limb Immobilization[END_REF][START_REF] Glover | Immobilization Induces Anabolic Resistance in Human Myofibrillar Protein Synthesis with Low and High Dose Amino Acid Infusion[END_REF], particularly related to aging [START_REF] Hvid | Effects of Ageing on Single Muscle Fibre Contractile Function Following Short-Term Immobilisation[END_REF][START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF][START_REF] Perkin | A Reduced Activity Model: A Relevant Tool for the Study of Ageing Muscle[END_REF][START_REF] Vigelso | Effects of Immobilization and Aerobic Training on Proteins Related to Intramuscular Substrate 138 Storage and Metabolism in Young and Older Men[END_REF], and identifying potential countermeasures [START_REF] Vigelso | Effects of Immobilization and Aerobic Training on Proteins Related to Intramuscular Substrate 138 Storage and Metabolism in Young and Older Men[END_REF][START_REF] Mitchell | Impact of Dairy Protein During Limb Immobilization and Recovery on Muscle Size and Protein Synthesis; a Randomized Controlled Trial[END_REF][START_REF] Edwards | High-Dose Leucine Supplementation Does Not Prevent Muscle Atrophy or Strength Loss over 7 Days of Immobilization in Healthy Young Males[END_REF].

Lower-limb immobilization models can be useful to investigate local muscle disuse since movements in the casted leg are tightly controlled during the protocol. Of importance for SB research, the lower-limb immobilization protocol likely induces increases in sedentary time. This can be presumed from free-living studies showing a higher sedentary time and lower PA during casting due to lower-or upper-limb fractures compared to healthy peers (100) or previous PA level (101). Similar to bed rest studies, immobilization protocols impose extreme changes in PA and SB levels. Additionally, the absence of control for PA level requires cautious interpretation of findings on systemic alterations in organs and systems other than the impacted limb.

Reduced daily step count

Reduced step count models have a focus on addressing how physical inactivity (reduced daily movement) is manifested in the daily lives of the majority of the population. For reference, median daily steps count in adult and older adult populations is typically around 5,000 (102). However, it is common that people may intermittently transition to lower daily step count. Reduced steps models aim to mimic such transitions by reducing participants' daily ambulatory activity from normal to sub-normal levels (103-106). Most protocols aim to reduce the total amount of daily steps to ~1,500 steps/day (range: 750 to 5,000) (105,(107)(108)(109)(110)(111)(112)(113). While the subsequent reduction in PA does not constitute complete disuse, it is plausible to assume that even short-term exposure to such periods of physical inactivity may have profound physiological consequences (109).

The main strength of reduced steps models is that they induce changes that are more similar to typical reductions in ambulatory activity that can occur in daily living due to hospitalization (740 to 2620 steps/day) (114, 115) as compared to strict bed rest, but it is less severe than those observed due to sickness (i.e., influenza-like symptoms; average of 924 steps/day) (116). The measure of PA level used in these studies has almost exclusively been step count. SB is not typically the focus and only a few have accurately reported increases in sedentary time during the step reduction protocols (106,112,113,117), with inferences made about changes in sedentary time as a consequence of reductions seen in time spent in ambulatory activities.

Reducing and interrupting prolonged sedentary behavior

Experimental models aimed at reducing and/or interrupting sitting time in laboratorybased and free-living settings are a relatively new approach. A key distinction here from the experimental models described above is the 'solution focused' treatment paradigm whereby typically physically inactive/sedentary individuals modify their typical low PA level to a higher PA level (118). Most reducing/interrupting prolonged SB models are acute in nature (most lasting >2 hours but <24 hours) (75-77, 119, 120). There is a growing number of multi-days (≤4 days) (121-124) to longer-term clinical trials (2 weeks to 36 months) [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF][START_REF] Shrestha | Effectiveness of Interventions for Reducing Non-Occupational Sedentary Behaviour in Adults and Older Adults: A Systematic Review and Meta-Analysis[END_REF].

Acute and multi-day protocols have utilized a control condition of imposed prolonged sitting and one or more experimental conditions involving, for example, a single continuous bout of activity and/or frequent, short bouts of activity (often referred to as 'breaks') [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. These experimental conditions may vary in terms of frequency, intensity, duration, and type of activity used to interrupt sitting [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF][START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. Acute models targeting reducing/interrupting prolonged SB can provide insights on the physiological effects and underlying mechanisms of such strategies. These studies have been typically conducted in highly controlled research environments, which allows accurate monitoring of potential confounders, including PA level. Consequently, the control condition (prolonged sitting) and experimental conditions (activity protocols) are typically unrepresentative of daily living activity patterns (125). To date, it is still uncertain whether some of the acute adaptations observed within this experimental model can be sustained over time [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF] and whether maladaptations to sitting are an impairment to, or a sign of plasticity of, the physiological systems; e.g., health being defined as the ability of our body to cope with daily-life challenges (i.e., phenotypic flexibility) (126).

Longer-term clinical trials have generally incorporated a control condition, in which sedentary/physically inactive participants are instructed to maintain their lifestyle, or have received usual care, and an experimental group, in which participants undergo an intervention to reduce/interrupt SB [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF][START_REF] Shrestha | Effectiveness of Interventions for Reducing Non-Occupational Sedentary Behaviour in Adults and Older Adults: A Systematic Review and Meta-Analysis[END_REF]. Longer-term randomized clinical trials can provide useful information on the longer-term dose-response effects of reducing/interrupting on health outcomes. Due to the nature of these studies, effectiveness of interventions at reducing/interrupting sedentary time, adherence to intervention, and duration, frequency and intensity of interruptions to SB must be closely monitored, as these factors likely affects the effects of such interventions on health outcomes.

Semantic considerations for interpreting the evidence that can be pertinent to sedentary behavior research

In this review, SB is defined based on both physiological and postural features (see subsection 2.1) and is being considered as a distinct behavior that coexists with physical inactivity in daily living. In studies where SB cannot be separated from physical inactivity due to methodological limitations, the evidence that we will now consider below will be discussed in light of study limitations and noted as a consequence of both behaviors. Given the interdependent nature of SB, LPA and MVPA and the lack of studies focusing on this interrelationship, changes in SB and PA level will be reported whenever original studies or meta-analyses reported such changes. This information will be particularly useful to triangulate available evidence to better understand potential differential, additive and/or interacting effects of behaviors on physiological outcomes.

The duration of SB interventions will also be reported as we address the relevant findings. The terms 'acute' and 'multi-days' will be used for studies lasting hours to ≤14 days. The term 'longer-term' will be used for studies lasting >2 weeks to years.

Adaptations to physical inactivity are not the opposite of adaptations to exercise/PA [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF][START_REF] Hamilton | Too Little Exercise and Too Much Sitting: Inactivity Physiology and the Need for New Recommendations on Sedentary Behavior[END_REF]127). Accordingly, evidence from studies using models for increasing SB will be discussed separately from those of models aimed at reducing/interrupting SB. The consequences of increasing SB will be discussed using evidence from bed rest, bed rest combined with exercise, immobilization/casting, reduced daily steps, and acute studies that included a condition imposing prolonged SB. In contrast, the effects of reducing/interrupting SB will be discussed using evidence from acute and longer-term studies that include at least one condition aimed at reducing/interrupting SB with multiple active bouts. Because of the limitations associated with bed rest models, evidence from bed rest related to whole-body outcomes will not be discussed herein.

However, given the absence of evidence from other human models of increased SB, bed rest data will be used to inform the potential mechanistic underpinnings of excessive and prolonged SB. Evidence from detraining, confinement, and natural experiments have not been used either, as study findings are constrained by limitations related to study design, targeted population (e.g., athletes, those highly physically active), and the lack of control for sedentary time.

Finally, SB research studies to date have included a variety of population groups.

Therefore, the use of some key terms has been standardized over the next sections to facilitate the discussion of available evidence. Adult and older adult groups have been defined according to age cut points used by the original studies ('adults': 18-59 or 18-64; 'older adults': ≥60 or ≥65 years old). The term 'children' has been used for 5-12 years old and 'adolescents' for 13-17 years old. With respect to health status, the term 'healthy' will be used to refer to population groups without any existing medical condition. Otherwise, health status or condition (e.g., overweigh, obesity, type 2 diabetes) will be reported along with study findings. The sex of participants will be reported for studies that included females or males only. Sex-neutral terms will be used in studies that included participants from both sexes. The considerations outlined above in Section 2 provide perspectives and caveats of relevance to the evidence we address below for each of the relevant major bodily systems and processes. Accordingly, in the following sections (3 to 8), we examine the relevant physiological evidence relating to: body weight and energy balance; intermediary metabolism; cardiovascular and respiratory systems; the musculoskeletal system; the central nervous system; and immunity and inflammatory responses. Fourteen days of reduced step count (from 10,501 to 1,344 steps/day) reduced leg lean mass (~0.5 kg) and increased intra-abdominal fat mass (7%), but not total fat mass in healthy male adults (108,109). Other studies (14 to 20 days; from ~11,500 to 2,000 steps/day) have shown similar alterations in healthy male adults, but also revealed increases in total and percent body fat (~3 to 14%) (106,107,128). Interestingly, alterations in body composition observed after 14 days of reduced step count (81% reduction from baseline plus a 3.7 h/day increase in sedentary time) returned to baseline levels after resuming habitual PA for 14 days in healthy adults with or without a first-degree relative with type 2 diabetes (112). Although inconsistent across studies, most step reduction protocols were detrimental for at least one body compositionrelated outcome in healthy older adults (105,110,129,130). Fourteen days of reduced step count (from ~9,000 to 3,000 steps/day) also resulted in intramuscular (nuclear/myofibrillar fraction) ceramides accumulation (~20%) in healthy older adults (130).

Reducing and interrupting sedentary behavior

Evidence from longer-term studies

A meta-analysis has analyzed data from longer-term studies investigating the effects of SB interventions conducted in free-living settings on adiposity outcomes. Intervention duration ranged between 2 weeks and 36 months, and the average change in total sedentary time was -28.6 min/day [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. There were small significant reductions in body weight (-0.6 kg), waist circumference (-0.7 cm) and percent body fat (-0.3%) in adults and older adults, but no changes have been reported in BMI, total body fat and total fatfree mass [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. Another meta-analysis investigating the effects of SB interventions (range: 6 to 24 weeks) on body composition demonstrated significant decreases in total sedentary time (-64 min/day) and increases in walking time (27 min/day), but no significant changes in time spent standing and in MVPA (134). Authors noted small significant decreases in percent body fat (-0.7%) and waist circumference (-1.5 In children, an 8-month, school-based intervention using height-adjustable desks in the classroom was ineffective at reducing classroom and total daily SB (137).

Consequently, no significant changes were observed on BMI z-score and waist circumference (137). However, other longitudinal studies demonstrated that when coupled with increases in PA, reducing SB prevents unhealthy weight gain. The socioecological French ICAPS (Intervention Centered on Adolescents' Physical activity and Sedentary behavior) study (138) concomitantly targeted PA and SB through a 4-year multi-level intervention that focused on the school and family of the children and the children themselves. Compared to the controls (no intervention), pupils who received the intervention were more active, less sedentary and gained less weight throughout the 4 year-study (138). Importantly, these changes in physical behavior and the prevention of weight gain were maintained 2.6 years after the end of the intervention with the highest efficacy in the most sedentary adolescents (139).

In summary, it is evident that findings are inconsistent across studies and meta- of daily hours in SB + 45 min/day increase in MVPA, respectively). This resulted in a significant decrease in BMI (-0.5 and -1.1 kg/m 2 , respectively), body weight (-2.3 and -3.4 kg, respectively) and total body fat (-1.0 and -1.4%, respectively). The addition of interruptions to SB did not result in greater improvements in body composition compared with exercise training only (140). No changes in body composition outcomes were observed in the group reducing SB only (140).

Another small-scale, 6-month pilot study examined the effects of a stepping protocol during TV commercial (brisk walking around the room during at least 90 min of TV programming at least 5 days/week) versus brisk walking for 30 min/day (at least 5 days/week) in adults with overweight/obesity (141). After 6 months of intervention, both protocols increased the number of daily steps and decreased time watching TV (2,994 vs 2,956 steps/day and -1.2 vs -1.4 h/day, respectively) as well as TV-related energy intake (-282 vs -517 kcals/day, respectively). Both interventions significantly reduced percent body fat (-1.0 vs -0.9%, respectively), waist circumference (-2.5 vs -1.6 cm, respectively) and hip circumference (-1.9 vs -1.2 cm, respectively) at the 6-month timepoint, but neither changed body weight and BMI (141). In contrast, 4 weeks of interventions aimed at reducing SB (-53 min/day in SB with no change in MVPA) or increasing MVPA to at least 30 min day (+16 min/day of MVPA with no change in SB) did not lower BMI and waist circumference in physically inactive adults with obesity (142).

Clinical significance

There is some evidence of small to trivial improvements in body mass and composition (body weight [-0.6 kg], waist circumference [-0.7 to -1.5 cm] and percent body fat [-0.3 to -0.7%]) associated with reducing/interrupting SB. Mixed results have also been reported across original studies and meta-analyses. Despite intervention effects being small and likely not clinically relevant, it is important to highlight that mean baseline BMI ranged between 25 and 30 kg/m 2 and study duration varied between 6 to 24 weeks in available meta-analyses [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]134). It has been suggested that the weight loss induced by 1-year exercise programs is more pronounced in those with existing obesity as compared to individuals with overweight (1.1 to 1.5 kg less than individuals with obesity) (143). It is not clear whether baseline BMI, other measures of adiposity and duration of intervention may have affected responses to reducing/interrupting SB interventions.

Reducing SB and increasing LPA without increasing engagement in MVPA/exercise does not seem to be an effective strategy to improve markers of adiposity as compared to traditional, continuous exercise (141,142). These findings indicate that reducing/interrupting SB with PA in higher intensities might be required to improve markers of adiposity in those with overweight/obesity. As recently reviewed thoroughly (144), reducing/interrupting SB is likely not effective at inducing weight loss, like exercise, but may prevent unhealthy weight gain. Future studies will also need to investigate whether changes in SB/LPA trigger spontaneous behavioral and physiological compensatory responses (e.g., decrease in activity and/or non-activity energy expenditures, increase in appetite and food intake) like those observed following the initiation of exercise training and thought to minimize the effect of exercise on body weight (145,146), as further discussed in the next topics.

Total energy expenditure

Increasing sedentary behavior

Evidence from multi-day studies

Participation in high levels of SB results in lower energy expenditure and PA levels. For example, 7 days of exposure to a highly sedentary condition in free-living (increased sedentary time and limited participation in PA of any intensity) significantly reduced energy expenditure (-15% in MET-hour/week estimated from a validated accelerometer) (147).

Evidence from longer-term studies

In a clinical study requiring physically active, but not trained, lean male adults to refrain from PA for 1 month, total daily energy expenditure decreased by 8% due to a drop in activity-related energy expenditure only (127).

Reducing and interrupting sedentary behavior

Evidence from acute studies

Experimental studies have demonstrated that both standing and ambulatory interruptions to prolonged sitting time increased energy expenditure in adults, as a function of the duration, intensity and modality used (148-150). Interestingly, the increased energy utilization in response to frequent 2-minute moderate-intensity walking interruptions to sitting was maintained for ~4 minutes after every walking bout in adults.

When repeated throughout the day, performing 28 minutes of interruptions to sitting resulted in ~70 minutes of elevated energy utilization over 7 hours (150).

Evidence from longer-term studies

It is still unclear whether SB reducing/interruption interventions may result in compensatory changes in total energy expenditure and/or energy balance over the longer term. A study demonstrating the differential impact of low-and moderateintensity training provides some relevant insights (151). Moderate-intensity aerobic exercise training (60% VO 2reserve ) tended to increase total energy expenditure in female adults with overweight/obesity compared to low-intensity training (40% VO 2reserve ) matched for energy expenditure over 3 months of intervention. Exercise energy expenditure was almost entirely compensated (96%). Interestingly, greater energy compensation was observed in the moderate-intensity group than in the low-intensity group (161% vs 49%) (151). Participants in the low-intensity group spent more time walking and less time lying down compared to the moderate-intensity group. These behavioral and energetic differences translated into about 1 kg weight gain in the moderate-intensity group versus 1 kg weight loss in the low-intensity group (151). The clinical significance of these findings is still unclear.

Energy intake and appetite

3.3.1. Increasing sedentary behavior 3.3.1.1. Evidence from acute studies Granados and colleagues (152) showed that 1 day of sitting decreased energy expenditure without a reduction in appetite in adults, suggesting this would favor a positive energy balance. This is consistent with findings that demonstrated no compensatory decline in ad libitum food intake in response to large reductions in energy expenditure (~24%) in healthy male adults (153). Another study (154) also demonstrated that energy intake during 1 day of decreased energy expenditure (275 steps/day) was comparable to energy intake during higher PA levels (equivalent to ~1.5 and 2.1 resting metabolic rate) in healthy male adults.

Evidence from longer-term studies

While there is no evidence on the impact of a reduced step count protocol on appetite regulation, 14 days of reduced step count (81% reduction from baseline) did not significantly alter fasting adiponectin and leptin levels in health male adults (108).

Acute experimental findings indicate that increasing SB can result in positive energy balance, yet caution is warranted when interpreting the clinical relevance of these findings, as acute changes in appetite may not affect weight control in the longerterm. Longer-term investigation into the effects of increasing SB on energy balance is warranted.

Potential mechanisms. Several hormones are involved in the regulation of appetite and feeding behavior. A study demonstrated a significant decrease in adiponectin levels (~21%; a hormone associated with increased sensation of hunger) in healthy male adults following 16 days of horizontal bed rest, but no changes in other appetite-regulating hormones (ghrelin, peptide YY [PYY], glucagon-like peptide 1 [GLP-1], and leptin) (155). In a 2-month HDBR study in females fasting leptin was negatively associated with the spontaneous decrease in energy intake, thus suggesting a relationship between PA, leptin and food intake [START_REF] Bergouignan | Regulation of Energy Balance During Long-Term Physical Inactivity Induced by Bed Rest with and without Exercise Training[END_REF].

Reducing interrupting sedentary behavior

3.3.2.1. Evidence from acute studies Over a 12-hour period, performing hourly 5-minute vigorous-intensity walking bouts (60-65% VO 2peak , total: 60 minutes) resulted in lower perceived hunger (~23%) in the midafternoon hours as compared to prolonged sitting and an energy-matched moderateintensity continuous walking bout (60-65% VO 2peak , total: 60 minutes) in adults with obesity, but did not affect PYY levels (156). Changes in perceived hunger were not observed for continuous exercise (60-65% VO 2peak , total: 60 minutes) followed by prolonged sitting (156). Another study demonstrated that, while frequent 5-minute moderate-walking interruptions (perceived effort: 12 -13 "somewhat hard", total: 30 minutes) did not affect hunger and desire for food consumption, this strategy resulted in lower food cravings (~6%) compared to prolonged sitting in healthy adults (157). This was not observed for continuous exercise (perceived effort: 12 -13 "somewhat hard", total: 30 minutes) followed by prolonged sitting (157). Furthermore, while interrupting sitting with 2-minute light or moderate-intensity walking bouts every 20 minutes (perceived effort: 6-9 "very light" and 12-14 "somewhat hard", respectively; total: 28 minutes) did not alter appetite, it resulted in reduced relative energy intake (39 and 120%, respectively) that was not compensated for in a subsequent meal in healthy adults, which could have important implications for weight management (158). In contrast, other studies showed no alterations in appetite measures, ad libitum intake and circulating gut hormone concentrations following interruptions to sitting in healthy adults (158-160). It is not clear whether type, intensity, and frequency of interruptions to sitting can differentially affect energy intake and appetite.

Evidence from longer-term studies

Replacing prolonged sitting time with regular standing bouts at the workplace (a 21% reduction of workplace sedentary time) reduced dietary intake (~10%) in sedentary, adult office workers after 4 weeks of intervention (161). It remains unclear the extent to which this reduction in caloric intake impacted measures of adiposity and other cardiometabolic outcomes. Longer-term investigation into the effects of reducing/interrupting SB on appetite and food intake is warranted.

INTERMEDIARY METABOLISM

Glucose metabolism

Increasing sedentary behavior

Evidence from acute and multi-day studies

Experimental findings show that in healthy adults as little as 1 day of exposure to SB (~17 h/day of SB) combined with energy surplus reduced whole-body insulin sensitivity (-39%), but did not change fasting glucose and insulin concentrations, as compared to a minimal sitting condition (~6 hours of sitting). Importantly, reducing energy intake to match energy demand during prolonged sitting significantly attenuated, but did not fully mitigate, the decline in insulin action (-18%) (162). This finding indicates that excessive sitting might be detrimental for insulin sensitivity irrespective of energy balance. Seven days of exposure to a highly sedentary condition (increased sedentary time and limited participation in PA of any intensity) did not alter fasting glucose and insulin concentrations in healthy-lean adults (147). Increasing SB significantly increased 2-hour post-load insulin concentration (38.8 uIU•mL -1 ) and reduced insulin sensitivity (-17.2%), as assessed by a composite insulin sensitivity index. Changes in time spent in prolonged sedentary bouts (>30 and 60 continuous minutes), but not in LPA and MVPA, were positively associated with 2-hour post-load insulin concentrations (147).

In a crossover randomized trial, healthy adults performed 10 days of reduced steps (from 12,154 to 4,275 steps/day, with a 10% increase in SB) while consuming a control diet (16% protein, 64% carbohydrate, 20% fat; 80% of daily energy need) or a high-protein diet (30% protein, 50% carbohydrate, 20% fat; 80% of daily energy need) (163). Independent of diet, there were no changes in fasting glucose and insulin concentrations, and post-load glucose and insulin responses in healthy adults following step reduction. However, in another study 10 days of step reduction resulted in reduced insulin sensitivity along with increases in carbohydrate oxidation measured in response to an OGTT [START_REF] Akins | Inactivity Induces Resistance to the Metabolic Benefits Following Acute Exercise[END_REF]. Combining step reduction with overfeeding also increased fasting glucose and insulin concentrations, post-load 2-hour glucose concentrations, and postload glucose responses in healthy male adults (163), thus suggesting that energy surplus exacerbates the metabolic deteriorations triggered by exposures to both SB and physical inactivity.

Evidence from longer-term studies

Fourteen days of reduced step count (from 10,501 to 1,344 steps/day) resulted in rapid decreases in whole-body (~58%) and peripheral insulin sensitivity (~17%) in healthy male adults (108, 109), which was accompanied by a significant reduction in insulinstimulated pAkt thr308 /total Akt protein expression (108). Similar alterations have been observed in healthy adults with/without a first-degree relative with type 2 diabetes (112) and older adults (130,164). Reducing daily step count (3 days, from 12,956 to 4,319 steps/day) increased postprandial (30 to 90 minutes after a meal) glucose responses ( 6to 9%) and glycemic variability (33 to 97%) in healthy adults as assessed by continuous glucose monitors, despite the absence of changes in post-load glucose responses following an oral glucose tolerance test (117). Twenty days of reduced steps (from 14,000 to 3,000 steps/day) increased total glucose oxidation in healthy physically active male adults, which was associated with a significant decrease in nonprotein respiratory quotient during an oral glucose tolerance test (106), which indicates the development of metabolic inflexibility (i.e., the inability of the body to adjust substrate use to changes in substrate availability). While no changes have been reported for fasting glucose concentrations following step reduction protocols in healthy adults (106)(107)(108)117), some studies demonstrated increases in fasting insulin concentrations (106,107,117). The absence of changes in glycemia may reflect compensatory increased insulin levels in response to reduced step count. Resuming habitual daily activities was sufficient to restore whole-body insulin sensitivity to baseline levels in healthy adults and older adults (107,112,165).

Potential mechanisms. Short-to longer-term exposures (3 to 90 days) to horizontal bed rest and HDBR are associated with reduced whole-body insulin sensitivity (166, 167), altered fuel selection towards the use of carbohydrate (166, 168), and metabolic inflexibility that precedes the development of glucose intolerance (168). Some mechanisms have been proposed including changes in body composition, body fat repartition, alterations in lipid content, oxidative stress, and capillary density (169).

While some have been confirmed, controversial results have been obtained for others.

For example, 60 days of HDBR induced fat accumulation in skeletal muscle (170) and with low-grade inflammation (171). However, Shur and colleagues (172) failed to demonstrate an increase in intramyocellular lipid content following 3 and 56 days of HDBR, which was not confounded by positive energy balance unlike previous studies (173,174). Similarly, after one week of bed rest, reduced insulin sensitivity was observed along with reduced oxidative capacity but not increases in muscle lipid level or degree of saturation, markers of oxidative stress or reductions in capillary density, suggesting other mechanisms are likely at play [START_REF] Dirks | One Week of Bed Rest Leads to Substantial Muscle Atrophy and Induces Whole-Body Insulin Resistance in the Absence of Skeletal Muscle Lipid Accumulation[END_REF]. Pronounced transcriptomic changes of the skeletal muscle metabolic pathways have also been proposed. Shur and colleagues (172) reported extensive changes in mRNA abundance in gene targets controlling carbohydrate metabolism (40 transcripts) after only 3 days of HDBR. These changes preceded the modifications in whole-body fuel selection and the reduction in muscle glycogen storage. Another study demonstrated similar alterations in the expression of genes associated with insulin resistance and fuel metabolism following 9 days of bed rest (175). Additionally, bed rest-induced reduction in insulin sensitivity has been accompanied by reduced skeletal muscle GLUT-4, hexokinase II, protein kinase B (Akt) 1, and Akt2 proteins content, and decreased insulin-stimulated glycogen synthase (GS) activity and Akt signaling (176). These findings suggest that both decreased glucose transport and decreased nonoxidative glucose metabolism in skeletal muscle contribute to changes in carbohydrate metabolism (FIGURE 3).

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---------------------------------INSERT FIGURE 3 ABOUT HERE ----------------------------------- 4.1.1.3. Clinical significance
Excessive SB has been positively associated with increased 2-hour post-load glucose and fasting insulin concentrations in the general population, independent of participation in MVPA (177,178). Experimental models imposing periods of increased sedentary time in both adults and older adults consistently induced alterations in whole-body and peripheral insulin sensitivity (-17 to -58%), but not in fasting glucose concentrations. The clinical significance of these findings is unclear. However, the dramatic reduction in insulin action within days of increased prolonged SB is relevant given that SB is the most prevalent behavior (8.3 to 11.5 h/day) (4) and alterations in markers of glucose control are associated with greater risk for cardiovascular disease and events even in those without diabetes (179, 180).

Reducing and interrupting sedentary behavior

Evidence from acute and multi-day studies

Acutely, interrupting sitting with frequent, short bouts of LPA improved postprandial glucose responses by 17.5% and insulin responses by 25.1% compared to prolonged, uninterrupted sitting in both healthy and metabolically impaired adults and older adults, as evidenced by meta-analysis [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF]. These results have been corroborated by other meta-analyses that have included studies investigating other types (e.g., standing still, walking, and simple resistance activities) and intensities (e.g., light, moderate and vigorous intensity) of interruptions to sitting (Cohen's d for glucose and insulin: -0.26 to -0.83 favoring intervention) [START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]181). However, it should be noted that some original studies have not demonstrated changes in postprandial glucose and/or insulin responses following at least one of the interruptions to SB protocols (standing, simple resistance activities, LPA to VPA walking) compared to prolonged sitting [START_REF] Peddie | The Effects of Prolonged Sitting, Prolonged Standing, and Activity Breaks on Vascular Function, and Postprandial Glucose and Insulin Responses: A Randomised Crossover Trial[END_REF]123,124,(182)(183)(184)(185)(186)(187)(188)(189)(190)(191)(192)(193)(194)(195)(196).

Reducing/interrupting sitting for 3 to 4 days reduced postprandial glucose (121,197) and insulin responses (122)(123)(124)197) in adults and older adults with overweight/obesity and type 2 diabetes (PA intensity and duration: 3.2 km/h, 2.8 METs, total: 34 min/day (121); perceived effort 13 "somewhat hard", total: 45 min/day (124); 93-95 steps/min, total: 3.1-4.1 h/day of standing and 3.1-4.9 h/day of stepping (122,123,197)). However, results have been more inconsistent for postprandial glucose responses following interruptions to sitting with standing/LPA (122, 123) and moderate PA (MPA) (124). No changes were observed in fasting glucose concentrations following 3 days of interrupting SB with standing or LPA (121)(122)(123)197) and MPA (124), but reductions in fasting insulin concentration were reported in adults with overweight (-1.8 mIU/L) (122) and type 2 diabetes (-13 pmol/L) (197). Despite these inconsistencies, insulin sensitivity improved after 3 days of interrupting SB (12-29%) (122-124, 197).

Interestingly, 3 days of interrupting prolonged sitting with regular 2-minute bouts of lightintensity walking (pace: 3.2 km/h, 2.8 METs, perceived effort: 6-11 "fairly light"; total: 28 minutes) sustained, but did not further enhance, improvements in postprandial glucose (-4%; estimated average concentration: 103 mg/dL following interruptions vs 107 mg/dL following prolonged sitting) and insulin responses (-12%; estimated average concentration: 262 pmol/L following interruptions vs 297 pmol/L following prolonged sitting) observed on the first day of intervention ( 121).

Both light-and moderate-intensity bouts improved postprandial glucose and insulin responses, while standing interruptions did not significantly affect these responses [START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF]181,198,199). A pooled analysis of three acute laboratory-based trials also showed that the estimated energy cost of interruptions to sitting was associated with lower postprandial glucose and insulin responses in a dose dependent manner in sedentary adults with overweight or obesity. Specifically, light-and moderate-walking interruptions (3.2 and 5.8 km/h; 2.8 and ~4.3 METs, respectively; 2-minute bouts every 20 minutes, total: 28 minutes), but not standing still, significantly reduced postprandial responses of both markers compared to prolonged sitting ( 200). Yet, a meta-analyses (181) and a few original studies (201-203) suggested standing interruptions are effective at improving glucose responses.

As for frequency of the active interruptions, results have been inconsistent across studies. Interrupting prolonged sitting with 6-minute bouts of simple resistance activities every 60 minutes (squatting, calf raises; total: 36 minutes) was more effective at decreasing postprandial glucose responses (-21%, estimated average concentration: 193 mg/dL following interruptions vs 203 mg/dL following prolonged sitting) in adults and older adults with type 2 diabetes compared to a higher frequency of interruptions (i.e., 3-minute bouts every 30 minutes, total: 39 minutes) and 8 hours of prolonged sitting (186), for which no differences were observed. Additionally, less-frequent interruptions to sitting acutely improved glycemic control in the 4-hour period following lunch, while more-frequent interruptions were likely more beneficial for nocturnal glucose control, as assessed by continuous glucose monitors over 22 hours (204). No differences were noted for mean glucose and other markers of glucose variability between interruptions and prolonged sitting (204). Other studies also showed similar inconsistencies for glucose and insulin responses across different frequencies of interruptions to SB compared to prolonged sitting (188,192,205). In addition, Duran and colleagues (206) suggested higher frequency and higher duration of light-walking interruptions to sitting (3.2 km/h, 2.8 METs; 5 minutes every 30 minutes, total: 70 minutes) should be considered when targeting improvements in glycemic responses in healthy adults and older adults. This strategy significantly attenuated postprandial glucose responses as compared to sedentary control (total: 8 hours), and conditions with lower frequency (every 60 minutes, total: 35 minutes) and lower duration (1 minute, total: ~7 or 14 minutes) of interruptions to sitting, for which no changes were observed (206). To date, available evidence is not sufficient to draw clear-cut conclusions on the dose-response relationship between the frequency of the active bouts to interrupt prolonged sitting and post-prandial glycemia and insulinemia, and further studies are needed.

Subgroup analyses in original studies showed that attenuations in the magnitude of reductions in postprandial glucose (122,199,207) and/or insulin (199) responses are more pronounced in females than in males following interruptions to sitting as compared to prolonged sitting, which was corroborated by meta-analytic evidence [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF].

Additionally, meta-regressions revealed that higher BMI is significantly associated with greater reductions in postprandial glucose and insulin responses following interruptions to sitting as compared to prolonged sitting [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. Pooled data from three randomized crossover trials showed that those with higher underlying levels of fasting insulin and insulin resistance may derive greater reductions in postprandial insulin responses from regularly interrupting prolonged sitting than their healthier counterparts. Similarly, those with poorer fasting glucose and β-cell function may derive greater reductions in postprandial glucose responses from performing walking interruptions to sitting (208).

These findings have been corroborated by subgroup analyses in two meta-analyses indicating that improvements on glycemia were more prominent in metabolically impaired adults and older adults compared to healthy counterparts [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. Moreover, McCarthy and colleagues (209) demonstrated that reductions in postprandial glucose responses were more pronounced in those with lower cardiorespiratory fitness (25 th and 50th centiles) following light-intensity walking interruptions to sitting (3.0 km/h, 2.0 METs, 5-minute bouts every 30 minutes, total: 1 hour) (209). Altogether these findings show that females, those with higher BMI, higher insulin resistance and lower cardiorespiratory fitness may derive greater benefit from interrupting sitting with respect to glucose metabolism.

Results have been mixed in children and adolescents. Interrupting sitting with 3minute bouts of moderate-intensity walking (heart rate at 80% of ventilatory threshold, total: 18 minutes) significantly reduced postprandial insulin responses (-21%; estimated average concentration: 92 mIU/dL following interruptions vs 117 mIU/dL following prolonged sitting), but not glucose responses, in children with overweight/obesity compared to 3 hours of prolonged sitting (210). Insulin sensitivity, as assessed by the Matsuda index, was also greater during the interruption protocol (17%) (210). In healthy-weight children, this same interruption protocol significantly reduced postprandial glucose (-7%; estimated average concentration: 105 mg/dL following interruptions vs 112 mg/dL following prolonged sitting) and insulin (-32%; estimated average concentration: 30 uU/mL following interruptions vs 45 uU/mL following prolonged sitting) responses compared to 3 hours of prolonged sitting (211).

Furthermore, in healthy children and adolescents, both light-intensity walking interruptions to sitting (30% VO 2peak , 2-minute every 20 minutes, total: 42 minutes) and interruptions combined with two 20-minute bouts of moderate-intensity walking did not affect postprandial glucose and insulin responses compared to 8 hours of prolonged sitting (212).

Reducing/interrupting SB vs continuous MVPA/exercise. Acute (8 to 9 hours) reductions in postprandial glucose responses were more pronounced following frequent light-walking interruptions to sitting (total: 30 -42 minutes) compared to a continuous bout of activity (total: 30 minutes) in healthy and at-risk adults and older adults (213-215). However, other studies did not observe such differences between patterns of activity (216-219). Frequent moderate-intensity interruptions were more effective at improving postprandial glucose and insulin responses than a single, continuous bout of moderate-intensity activity when compared to prolonged sitting respectively), as evidenced by meta-analysis (198).

Another meta-analysis demonstrated similar results for postprandial glucose responses, but no differences were observed for insulin responses [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. In multi-day studies (2 to 4 days), reducing/interrupting SB was shown to be more effective at reducing fasting insulin, 24-hour glucose responses and duration of hyperglycemia episodes, but not other markers of glucose metabolism (e.g., fasting glucose), compared to continuous exercise in adults and older adults with type 2 diabetes (93 steps/min, total: 4.1 h/day of standing and 3.1 h/day of stepping vs 50-60% W max , total: 1 h/day) (197). In healthy adults and adults with overweight/obesity, there were no differences between patterns of activity in markers of glucose metabolism (124,220).

Potential mechanisms. The effects of interrupting sedentary time on glucose control are likely related to a greater reliance upon carbohydrate oxidation as fuel. Acute studies (6 to 9 hours) in healthy adults (214,218) showed that the lower glucose response following light-(25% VO 2max , total: 3.5 hours) and moderate-(~46% VO 2max , total: 30 minutes) walking interruptions to sitting compared to prolonged sitting (214, 218) was associated greater total carbohydrate oxidation. De Jong and colleagues (124) also demonstrated in adults with overweight or obesity that hourly 5-minute bouts of moderate-walking interruptions during sitting (perceived effort 13 "somewhat hard", total: 45 min/day) primarily relies upon carbohydrate as fuel over 24 h after 4 days of intervention as compared to prolonged sitting and a time-matched continuous bout of brisk walking. This effect does not appear to be related to energy expenditure and balance, but rather to increasing the frequency of muscle contractions spread across the day. However, these differences in substrate use were not accompanied by changes in fasting and postprandial glucose responses (124).

The skeletal muscle is the largest glucose consuming organ of the body ( 221 2-minute bouts of walking every 20 minutes, total: 28 minutes) further resulted in an upregulation of the contraction-stimulated glucose uptake pathway (i.e., adenosine monophosphate-activated protein kinase (AMPK)-mediated), while three consecutive days of interrupting sitting resulted in a transition towards upregulation of the insulinmediated glucose uptake pathway (i.e., Akt-mediated) along with greater capacity for glycogen synthesis (i.e., increase in total GSK3β protein expression) in the skeletal muscle of adults with overweight/obesity (226). In contrast, there were no differences in pAS160 Thr642 /AS160 ratio and GLUT4 protein expression in the skeletal muscle of postmenopausal females with rheumatoid arthritis following light-intensity walking interruptions to sitting (~25% heart rate reserve, 3-minute bouts every 30 minutes, total: 42 minutes) compared to 8 hours of prolonged sitting (213). Finally, although acute exposure to moderate-intensity active interruptions in adults with overweight/obesity was associated with increased gene expression of complex V of the electron transport chain indicating greater capacity for ATP production (226), four days of moderatewalking interruptions to sitting (perceived effort: 12-13 "somewhat hard", 5-minute bouts every hour, total: 45 minutes/day) did not elicit changes in mitochondrial respiration in presence of carbohydrates (227); of note this later measurement was performed in fasting state and >12 hours after the last active interruption. Notably, pathways associated with muscle contraction transcription signaling, namely oxidative phosphorylation and sirtuin signaling expression, were enhanced in the skeletal muscle of these same participants when compared to a highly sedentary condition, as indicated by pathway enrichment analysis with RNA sequencing data (227).

In the subcutaneous abdominal adipose tissue, interrupting prolonged sitting with light-intensity walking (3.2 km/h, 2.8 METs, 2-minute bouts of walking every 20 minutes, total: 28 minutes) led to a downregulation of pathways linked to carbohydrate oxidation and upregulation of pathways linked to lipid oxidation in adults with overweight/obesity as compared to prolonged sitting and moderate-intensity walking interruptions. In contrast, genes associated with glucose oxidation were upregulated in the moderateintensity walking condition (5.8 km/h, ~4.3 METs, 2-minute bouts of every 20 minutes, total: 28 minutes) (228).

These studies provided the first insights into the muscle regulatory systems and molecular processes underlying the effects of interrupting prolonged sitting on glucose metabolism (FIGURE 4).

--------------------------------INSERT FIGURE 4 ABOUT HERE --------------------------------4.1.2.2. Evidence from longer-term studies A meta-analysis examined findings from longer-term studies investigating the effects of SB interventions conducted in free-living settings on glucose/insulin outcomes.

Intervention duration ranged between 2 weeks and 36 months, and the average change in total sedentary time was -28.6 min/day [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. There was a small significant decrease in fasting insulin concentration (-1.42 pM), but no changes in fasting glucose concentration and HbA1c [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. Another meta-analysis investigating the effects of SB interventions (range: 6 to 24 weeks) on markers of glucose metabolism demonstrated significant decreases in total sedentary time (-64 min/day) and increases in walking time (27 min/day), but no significant changes in time spent standing and in MVPA (134).

Authors noted a small significant decrease in HbA1c (-0.2%), but no change in fasting glucose concentration in individuals with overweight, obesity, type 2 diabetes, cardiovascular, neurological/cognitive, or musculoskeletal diseases (134). A metaanalysis of studies aiming to replace SB with standing time (mean follow-up: 3.8 months) demonstrated a significant increase in total standing time (1.3 h/day) in adults along with significant decreases in fasting glucose (-2.53 mg/dL) and insulin (-2.30 mg/dL) concentrations (135). A systematic review of studies implementing workplace SB interventions in apparently healthy and overweight/obese desk-based office workers found that the effects of workplace interventions on markers of glucose metabolism have been inconsistent across studies. Only 6 out of 15 studies reported improvements in at least one glucose metabolism-related outcomes, while most studies showed no changes (136). Furthermore, three weeks of frequent active interruptions to sitting (increase of 744 steps/day from baseline, with no changes reported for SB) reduced mean fasting glucose levels (-0.34 mmol/L) and glucose variability (-2%) in highly sedentary adults with obesity, but did not improve glucose tolerance, insulin sensitivity, post-load glucose responses, and average interstitial glucose concentrations (229).

Altogether, it becomes evident that results on the effects of reducing/interrupting SB on markers of glucose metabolism are mostly inconsistent across studies and meta-

Clinical significance

There is some evidence of small improvements on markers of glucose control (fasting insulin [-1.42 pM], glucose [-2.53 mg/dL] and HbA1c [-0.2%] concentrations) associated with reducing/interrupting SB. However, mixed results have also been reported across original studies and meta-analyses. It is important to highlight that mean baseline levels of fasting glucose, insulin and HbA1c were mostly within normal ranges in available meta-analyses [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]134,135), which may partly explain the small magnitude of reported changes following SB interventions. In fact, these small changes are comparable to the findings that have been observed with continuous exercise in people without glycemic dysfunction (230).

The most commonly used marker of glucose control in clinical practice is HbA1c.

A reduction of 0.5 to 1.0% is generally used as a cut point for clinically meaningful change, which associates with significant reductions in risk of all-cause mortality, myocardial infarction, stroke, and heart failure in patients with type 2 diabetes (231).

One meta-analysis of SB interventions demonstrated a 0.2% reduction in HbA1c (mean baseline level was 6.4%) after 6 to 24 weeks of follow up (134), which is not clinically meaningful. Interestingly, a 3-year intervention (1-month exercise counseling every year; SB: -0.8 h/day, LPA: 0.7 h/day, and MVPA: 3 min/day) significantly reduced HbA1c values (-0.5%) in those with type 2 diabetes compared to standard care (i.e., general physician recommendations for increasing daily PA and decreasing SB) (232).

The highest quartile for changes in SB (-1.53 h/day and +10.5 min/day of MVPA) significantly reduced HbA1c values (-0.85%) as compared to other quartiles, with SB being an independent predictor of changes in HbA1c (233). Another randomized controlled trial aiming at increasing participation in standing and LPA (SB: -39 min/day, standing: 39 min/day) performed a subgroup analysis in the participants who had high fasting glucose concentrations at baseline and found effects sizes to be larger for fasting glucose and HbA1c (-7.2 mg/dL and -0.3%, respectively) (234). Altogether, these findings indicate that reducing/interrupting SB has the potential to improve glucose control in those with dysglycemia and type 2 diabetes.

Lipid metabolism

Increasing sedentary behavior

Evidence from acute and multi-day studies

Four days of exposure to a highly sedentary regime (14 hours sitting/day) did not alter fasting blood lipids concentrations (i.e., total cholesterol, HDL, LDL, and triglycerides) in healthy sedentary adults (123). Similar results were observed in those with overweight and obesity (122) and type 2 diabetes (197). Seven days of exposure to a highly sedentary condition (increased sedentary time and limited participation in PA of any intensity) did not alter fasting blood lipids concentrations in healthy-lean adults (147).

Evidence from longer-term studies

Fourteen days of reduced step count (from 10,501 to 1,344 steps/day) led to increased triglyceride responses (~21%) to an oral fat tolerance test in healthy male adults (109). A 14-day period of reduced steps (by 81%, and a 3.7 h/day increase in sedentary time) significantly increased fasting total cholesterol, LDL and triglycerides (by 0.5, 0.3 and 0.5 mmol/L, respectively) in healthy adults with/without a first-degree relative diagnosed with type 2 diabetes. These changes returned to baseline levels after resuming habitual PA for 14 days (112). Twenty days of reduced steps (from 14,000 to 3,000 steps/day) not only worsened fasting HDL concentration but decreased total lipid oxidation and increased fasting triglycerides concentration (0.3 mmol/L) along with de novo lipogenesis in healthy physically active male adults (106). Periods of reduced step count (14 days; from 9,008 to 2,994 steps/day) also resulted in increased serum and intramuscular ceramides in healthy older adults (130). Other studies demonstrated deleterious effects of reduced steps on blood lipids in healthy male adults (128, 235); however, results have also been inconsistent, with some studies demonstrating no effects (107,108,236).

Potential mechanisms. Results from bed rest studies have been inconsistent for blood lipids concentrations in healthy adults and older adults, with some reporting hypertriglyceridemia and hypercholesterolemia and others no effect [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]166,237). Bed rest also impairs lipid oxidation in both fasting and postprandial states, which was observed to be independent of detectable changes in energy balance (166) (FIGURE 3). This reduced fatty acid oxidation does not seem to be due to an impaired trafficking towards peripheral tissue but rather to structural, metabolic, and molecular changes in the skeletal muscle (170). For example, physical inactivity coupled with sedentariness has been shown to reduce content of slow oxidative muscle fibers (238-240), reduce mitochondrial density and oxidative capacity (241), and decrease the expression of genes involved in mitochondrial function (242). Gene expression and activity of enzymes coupled with oxidative metabolism, such as skeletal muscle LPL, fatty acid transporter into the myocyte (CD36) and into the mitochondria (carnitine palmitoyltransferase I [CPT1b]) (127,170), are also decreased (170,242,243). These changes are particularly relevant following meal ingestion since they lead to decreased clearance of dietary lipids, which can contribute to hyperlipidemia. In this line, decreases in the oxidation of dietary saturated, but not monounsaturated, fatty acids were observed (127,170,244) showed that 42 days of HDBR altered transcriptome signatures with up-regulation of genes in lipid metabolisms in liver samples, revealing mildly disturbed fatty acid metabolism (251). In humans, indices of fatty liver such as high levels of plasma transaminases (252) have been associated with increased de novo lipogenesis (106), an index of hepatic insulin resistance.

Clinical significance

Excessive SB has been positively associated with increased fasting triglyceride concentrations and decreased fasting HDL concentrations in the general population, independent of participation in MVPA (253-255). Some detrimental changes in blood lipids have been reported in reduced step count studies in both adults and older adults, but results have been inconsistent. The clinical significance of these findings is unclear.

Reducing and interrupting sedentary behavior

Evidence from acute and multi-day studies

Findings from single and multi-day experimental studies examining the effects of interrupting prolonged sitting on fasting (122,123,147,197) and postprandial lipid concentrations [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF] have been less consistent than those observed for glucose and insulin responses. Some original studies failed to show improvements in triglycerides following at least one of the interruptions to SB compared to prolonged sitting (121,124,186,188,190,191,201,207,213,214,256). Two meta-analyses [START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF] indicated no effect of standing, light-and moderate-intensity activity interruptions to sitting on postprandial triglycerides responses in healthy and metabolically impaired adults and older adults compared to prolonged, uninterrupted sitting. In contrast, one meta-analysis (77) indicated a small significant effect (Cohen's d: -0.26 favoring intervention) of interruptions to sitting on postprandial triglycerides in healthy and metabolically impaired adults and older adults compared to prolonged, uninterrupted sitting. This was mainly driven by the evidence from multi-day studies, which may suggest an additive effect of interruptions to sitting on blood lipids [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF].

The magnitude of changes in triglyceride responses following interruptions to sitting have been reported to be independent of the intensity of PA interruptions (standing vs light-vs moderate-intensity interruptions), as indicated by subgroup analysis [START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF]. For frequency of interruptions to sitting, there were no differences in postprandial triglycerides responses following light-intensity walking interruptions to sitting every 20, 60 and 120 minutes (30% VO 2max , total: 48 minutes) in male adults with central obesity (188). Similarly, there were no differences between low-and highfrequency simple resistance activities interruptions to sitting (squatting, calf raises, total: 36 minutes; or moderate-intensity walking, i.e., 65% VO 2peak , total: 30 minutes) on postprandial triglycerides responses in adults and older adults with type 2 diabetes (186) and in healthy female adults (192). It is unclear whether the type of interruptions to sitting differentially affect markers of lipid metabolism.

The magnitude of changes in triglyceride responses following interruptions to sitting were not affected by meal composition, BMI, and population characteristics, as indicated by subgroup analysis and meta-regressions [START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]. Interestingly, fasting triglycerides had a negative quadratic association with postprandial triglyceride responses to a single, moderate-walking bout followed by intermittent light-walking interruptions compared to prolonged sitting in healthy older adults. This may imply that those with high triglyceride levels were more resistant to intervention-induced reductions in triglycerides responses (378).

In children with normal weight or overweight/obesity, interrupting sitting with 3minute bouts of moderate-intensity walking (heart rate at 80% of ventilatory threshold, total: 18 minutes) did not affect postprandial triglycerides and free fatty acids responses compared to 3 hours of prolonged sitting (210,211). Similarly, light-intensity walking interruptions to sitting (30% VO 2peak , 2-minute every 20 minutes, total: 42 minutes) did not affect postprandial triglyceride, HDL and LDL responses in healthy children and adolescents compared to 8 hours of prolonged sitting (212).

Regarding other markers of lipid metabolism, acute and multi-day studies ( 6hours to up to 4 days) indicate no changes in postprandial concentrations of free fatty acids (124,215,217,218,220,257) Reducing/interrupting SB vs continuous MVPA/exercise. Acutely (7 to 9 hours), a continuous bout of PA (moderate intensity, total: 30 minutes) in the morning was more effective at attenuating postprandial triglyceride responses than frequent standing (45minute interruptions every 15 minutes, total: 4.5 hours) (257), moderate-walking (46% VO 2max , 1.6-minute interruptions every 28 minutes, total: 30 min) (214) and vigorouscycling (70% VO 2max , 6-minute interruptions every 40 minutes, total: 30 minutes) (258) interruptions to sitting in healthy adults. In contrast, other studies did not report such differences between patterns of activity (213,215,216,218) or showed a superiority of interruptions to sitting at decreasing postprandial triglyceride responses in healthy and at-risk adults and older adults (217, 219). A meta-analysis summarizing acute evidence demonstrated no differences between interruptions to sitting and a continuous bout of activity on postprandial triglycerides responses [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF], but activity protocols were not always matched for duration and energy expenditure. In multi-day studies (2 to 4 days), reducing/interrupting SB was shown to be as effective as continuous exercise in reducing fasting triglyceride concentrations in adults and older adults with type 2 diabetes (197). In healthy adults and adults with overweight and obesity, there were no differences between interruptions to sitting and continuous activity in postprandial triglycerides and free fatty acids responses (124,220).

Potential mechanisms. As expected, a single continuous bout of PA leads to increases in total fat oxidation compared to a sedentary control, while greater reliance upon carbohydrate was shown following interruptions to sitting in healthy adults and adults with overweight/obesity (124,214). Peddie and colleagues (214) observed a significant reduction in postprandial triglyceride responses following a single moderateintensity walking bout (~46% VO 2max , total: 30 minutes) compared to prolonged sitting and interruptions to sitting (214,218). Despite changes in substrate use, De Jong and colleagues (124) failed to detect reductions in fasting and postprandial triglycerides responses following a continuous bout of activity (perceived effort 13 "somewhat hard", total: 45 min/day). It is likely that glycogen storage was partly depleted when performing continuous PA, thus allowing lipids to be oxidized for energy expenditure and glycogen pools to be refilled, which likely did not occur during brief interruptions to sitting as glucose was constantly available and competing against lipids. Additionally, postprandial responses of markers for hepatic fatty acid oxidation (total ketone bodies and acetoacetic acid) were elevated following continuous exercise, but not interruptions to sitting. This indicates increased hepatic fatty acid oxidation and reduced availability of triglycerides for incorporation into VLDL with exercise (215).

It is difficult to explain the inconsistencies in the acute effects of reducing/interrupting SB on postprandial triglyceride responses. Based on findings from animal models, it has been hypothesized that the lack of muscle contraction during exposures to increased SB reduces skeletal muscle LPL mass and activity and alters lipid metabolism [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF]. Of note, these defects were reversible only with light-intensity contractile activity but not MVPA/exercise [START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF][START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF]. No change was observed in serum inactive monomeric LPL protein concentrations following standing interruptions to sitting in healthy males (257) and in plasma pre-heparin LPL concentrations following walking interruptions to sitting in postmenopausal females (215). Similarly, no changes in LPL gene expression were reported following frequent light-to moderate-intensity walking interruptions to sitting (over 5 to 8 hours, 2-3-minute bouts every 20-30 minutes) in the skeletal muscle of adults with overweight/obesity (225) and postmenopausal females with rheumatoid arthritis (213). One possibility is that the pattern and frequency of activity used in published studies were insufficient to elicit changes in LPL enzymatic activity.

Evidence from longer-term studies

A meta-analysis examined data from longer-term studies of the effects of SB interventions conducted in free-living settings on blood-lipid outcomes. Intervention duration ranged between 2 weeks and 36 months, and the average change in total sedentary time was -28.6 min/day [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. There was a small significant increase in HDL (0.04 mM), but no change in total cholesterol, LDL and triglycerides, in response to interrupting/reducing SB interventions [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. Another meta-analysis investigating the

Clinical significance

There is some evidence of small improvements, likely not clinically meaningful, on fasting HDL (0.04 mM) concentrations associated with reducing/interrupting SB. However, no consistent improvements have been reported for other blood lipids. It is important to highlight that those studies within available meta-analyses reported mean baseline values for all fasting blood lipids within normal ranges [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]134), which may contribute to the small magnitude of changes reported following SB interventions.

Putting these results into perspective with the effects of continuous exercise, the small benefit for HDL is comparable to the increases in HDL associated with supervised aerobic exercise in adults and older adults with type 2 diabetes (259). A meta-analysis also demonstrated continuous aerobic exercise decreased fasting triglycerides concentrations (-6.8 mg/dL), but not total cholesterol, LDL and HDL, in adults and older adults with overweight/obesity (260). The current evidence indicates that reducing/interrupting SB has marginal effects on fasting blood lipids. Longer-term investigations into the effects of reducing/interrupting SB on blood lipids is warranted, particularly in individuals with dyslipidemia. Step reduction (~80% reduction from baseline) for 14 days resulted in a significant decrease in postprandial myofibrillar protein synthetic rate (-25 to -50%) in older adults (105,110). This also seems to be the case in clinical populations, in which reducing step count [START_REF] De | Original Research Socio-Demographic Patterning of Self-Reported Physical Activity and Sitting Time in Latin American Countries: Findings from ELANS[END_REF]362 to 991 steps/day) significantly reduced measures of the 14day integrated rates of muscle protein synthesis (~-12%) in older adults with overweight and prediabetes, which remained lower after resuming habitual activity for 14 days (164). Using a unilateral leg model, Devries and colleagues (105) demonstrated that the step reduction-induced decrease in postprandial myofibrillar protein synthetic rate can be fully mitigated in older adults by performing unilateral resistance exercise training (3 times/week) while performing step reduction. A study employing 14 days of unilateral immobilization also showed reduced post-absorptive muscle protein synthesis (-27%) in healthy adults [START_REF] Glover | Immobilization Induces Anabolic Resistance in Human Myofibrillar Protein Synthesis with Low and High Dose Amino Acid Infusion[END_REF]. Potential mechanisms. Bed rest studies indicate the loss of body protein with physical inactivity/SB is predominantly due to a decrease in muscle protein synthesis (265). This latter was associated with reductions in lean mass (-1.7%) following 14 days of HDBR (265).

Clinical significance

The pronounced reductions in muscle protein synthesis (-12 to 50%) following periods of increased SB are likely relevant. The physiological and clinical impact of increasing SB on skeletal muscle will be further discussed in topic 6.1.

Reducing and interrupting sedentary behavior

Evidence from acute studies

Acutely, myofibrillar protein synthesis was greater following light-walking (1.9 km/hour, <2.8 METs, 2 minutes of walking every 30 minutes, total: 30 minutes) and squatting interruptions to sitting (15 body-weight squats every 30 minutes, total: 225 repetitions) as compared to 7.5 hours of prolonged sitting in healthy adults (~47% and ~20%, respectively) (266). The increases in myofibrillar protein synthesis are comparable to those observed following an acute bout of structured resistance exercise (~40%) in healthy male adults (267). Additionally, rpS6 Ser240/244 phosphorylation was greater in squatting interruptions compared to prolonged sitting, but no differences were observed for other anabolic signaling protein targets (4E-BP1 Thr37/46 , eEF2 Thr56 , mTOR Ser2448 , ERK1/2 Thr202/Tyr204 ) (266). This could suggest that the squats interruptions resulted in the stimulation of translation initiation and myofibrillar protein synthesis. In contrast, walking had no effect on any anabolic signaling protein targets, which may be related to a lower activation of the vastus lateralis during walking compared to squatting (268). It is not clear whether type, intensity, and frequency of interruptions to sitting differentially affect markers of protein metabolism.

As for whole-body nutrient oxidation, De Jong and colleagues (124) showed that frequent 5-minute bouts of brisk walking at every hour (total: 45 min/day) increased protein oxidation (11.4%) during the sleeping period (8 hours) as compared to the sedentary condition (67% of waking hours) in people with overweight/obesity. The authors suggest that the greater disappearance in protein may reflect the use of protein for gluconeogenesis to replenish muscle glycogen, as the short bouts of activity likely triggered the use and replenishment of glycogen stores, thus enhancing glycogen turnover (124).

It has been proposed that muscle hypertrophy following resistance exercise occurs as a result of summed periods of repeated acute exercise-induced positive protein balance where muscle protein synthesis exceeds muscle protein breakdown (269). As such, the practical relevance of acute increases in myofibrillar protein synthesis (20-47%) seen following aerobic and resistance activity interruptions to sitting and the extension of skeletal muscle remodeling should be addressed in future longerterm studies.

CARDIOVASCULAR AND RESPIRATORY SYSTEMS

Hemodynamics

Increasing sedentary behavior

Evidence from acute studies

A seated posture creates bends in major blood vessels, such as the femoral and popliteal arteries, which may result in turbulent blood flow patterns (270,271). Also, such posture not only results in diminished skeletal muscle contractile activity that aids in venous return via the muscle pump, but also detrimentally affects blood flow and vascular shear stress (physiological stressors that may underlie the health benefits of PA on the endothelium) [START_REF] Dempsey | Sitting Less and Moving More: Implications for Hypertension[END_REF]. In healthy adults, shear rate in the lower limbs, but not in the upper limbs, is reduced after only 30 minutes of uninterrupted sitting [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]272). After approximately 2 hours, blood pools in the calf and whole-blood leg viscosity is also reduced (273). After 3 hours, blood flow in lower limbs is decreased in parallel with a further reduction in shear rate, as evidenced by meta-analysis [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]. Although most of the evidence suggests that prolonged sitting detrimentally affects peripheral hemodynamics (274), some studies have demonstrated no alterations in blood flow (275) and retrograde shear rate (276, 277) in healthy males following 3-5 hours of uninterrupted sitting.

A meta-analysis reported that exposures to prolonged, uninterrupted sitting resulted in significant increases in systolic blood pressure (3.2 mmHg) and mean arterial pressure (3.3 mmHg) among adults and older adults, but diastolic blood pressure was unaffected (119). In contrast, another meta-analysis demonstrated no change in mean arterial pressure following exposures to prolonged sitting >3 hours [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]. Some studies also failed to observe sitting-induced alterations in systolic (190,(278)(279)(280)(281), diastolic pressure (195,278,(282)(283)(284), and mean arterial pressure (281,(285)(286)(287)(288). In addition, 7 days of reduced step count (~9,000 to ~6,000 steps/day) did not modify systolic and diastolic blood pressure and mean arterial pressure in healthy-active adults (289).

Five days of reduced steps (from ~12,000 to 4,000 steps/day) did not affect femoral or brachial artery blood flow responses (104), nor did it alter shear rate in healthy-active male adults (103).

Changes in catecholamines have been inconsistent across studies; some indicated no changes and others small increases in plasma/serum concentrations in adults and older adults following 7-8 hours of prolonged sitting (284,290,291).

Increased lower leg and foot venous pressure/swelling was also observed after prolonged sitting, possibly impairing the regulation of capillary fluid filtration and edema formation in the feet (292).

Potential mechanisms. Atherosclerotic plaques have been reported to commonly manifest near arterial bifurcations, which is at least partially due to an unfavorable local hemodynamic environment (293). The seated position may mimic a similar environment to arterial bifurcations due to the 'bent artery' morphology created by 90-degree angles in the hip and knees. Acute evidence revealed that as little as 3 hours of leg bending, similar to sitting, produces detrimental hemodynamic changes in the popliteal artery as compared with a straight limb (294). Therefore, the 'bent artery' position induced by sitting may be a key contributor of changes in hemodynamics (274).

Evidence from longer-term studies

Unilateral leg immobilization (12 days) did not induce changes in blood pressure, mean blood flow in carotid, femoral and popliteal arteries in the immobilized leg versus the non-immobilized leg in healthy adults (295). However, mean blood velocity (~22%) and vessel shear rate (~35%) in femoral artery were increased in the immobilized leg (295).

Hemodynamic adaptations to lower-limb immobilization seem to be constrained to peripheral arteries, not altering large central arteries (295).

Fourteen days of step reduction (~82% reduction from baseline combined or not with unilateral low-load resistance exercise training, 3 times/week) did not alter superficial femoral artery blood flow during both fasted and fed state in healthy older males (105). Systolic blood pressure increased by 4 mmHg following a 14-day step reduction protocol (81% reduction from baseline; +3.7 h/day in sedentary time) in healthy adults with/without a first-degree relative with type 2 diabetes but returned to baseline after resuming habitual activity for 14 days (112). Diastolic blood pressure remained unchanged during step reduction and recovery periods (112).

Clinical significance

Acute exposures to prolonged sitting resulted in significant increases in systolic blood pressure (3.2 mmHg) and mean arterial pressure (3.3 mmHg) among adults and older adults (119). Similar increases (4 mmHg) were reported following 14 days of reduced step count in healthy adults with/without a first-degree relative with type 2 diabetes (112). While it is unclear whether these detrimental changes are sustained over time, the magnitude of these increases in blood pressure are likely to be clinically significant if maintained. For some perspective, at population level, 1 to 10 mmHg increases in mean systolic blood pressure are associated with increases in cardiovascular diseases incidence (296) and mortality (296, 297) and stroke mortality (298).

Reducing and interrupting sedentary behavior

Evidence from acute studies

A meta-analysis of acute randomized crossover trials showed that interruptions to sitting (aerobic, simple resistance activities and standing) reduced systolic blood pressure by 4.4 mmHg and diastolic blood pressure by 2.4 mmHg versus prolonged sitting (119). A meta-analysis demonstrated no change in systolic and diastolic blood pressure following standing interruptions to sitting compared to prolonged sitting in adults and older adults (181), suggesting interruptions at higher intensities may be necessary to affect blood pressure. However, mixed results have been reported by original studies. A systematic review summarized findings from acute randomized crossover studies investigating blood pressure responses to different intensities of interruptions to sitting in adults and older adults at risk for type 2 diabetes (299). Five out of ten studies found significant improvements in blood pressure responses following light-intensity interruptions, and three out of five studies demonstrated reductions following moderateto-vigorous-intensity interruptions (299). In contrast, only one out of six studies found reductions in blood pressure following standing interruptions to prolonged sitting (299), indicating that standing interruptions might be less effective at improving blood pressure responses.

Regarding the type of interruptions to sitting, the decreases in systolic blood pressure were mainly driven by studies implementing aerobic activity interruption strategies, as evidenced by a meta-analysis (119). In terms of frequency, interrupting prolonged sitting with 3-minute or 6-minute bouts of simple resistance activities every 30 or 60 minutes (squatting, calf raises; total: 36-39 minutes) did not elicit changes in blood pressure responses compared to 8 hours of prolonged sitting in adults and older adults with type 2 diabetes (283). Similarly, there were no differences in blood pressure responses following high (2-minute bouts every 20 minutes, total: 30 minutes) and low frequency (10-minute bouts every 60 minutes, total: 50 minutes) of standing bouts compared to 6 hours of prolonged sitting in older adults (195). In addition, Duran and colleagues (206) suggested light-walking interruptions to sitting (3.2 km/h, 2.8 METs) of high and low frequency (every 30 and 60 minutes, respectively) and high and low duration (5-and 1-minute interruptions, respectively) can be considered when targeting improvements in blood pressure responses in adults and older adults. These strategies significantly attenuated systolic blood pressure responses (-3 to -5 mmHg) as compared to sedentary control (total: 8 hours), and there were no differences between high/low frequencies and durations of interruptions to sitting protocols (206). Both high and low frequencies of single resistance activity interruptions (squatting, calf raises; every 30 vs 60 minutes, total: 36-39 minutes) significantly increased blood flow (125 and 114%, respectively) and shear rate (87 and 98%, respectively) in adults and older adults with type 2 diabetes as compared to 8 hours of prolonged sitting (283).

Reductions in systolic and diastolic blood pressure responses were more pronounced in older females than older males in response to a 30-min bout of exercise followed by frequent, brief light-intensity walking bouts as compared to 8 hours of uninterrupted sitting (291). This finding suggests that females might benefit more from performing exercise plus interruption to sitting strategy.

In preadolescent children, interrupting sitting with light-, moderate-and highintensity walking [START_REF] Dempsey | Sitting Less and Moving More: Improved Glycaemic Control for Type 2 Diabetes Prevention and Management[END_REF][START_REF] Kump | Sustained Rise in Triacylglycerol Synthesis and Increased Epididymal Fat Mass When Rats Cease Voluntary Wheel Running[END_REF] and 75% heart rate reserve, respectively; 2-minute bouts every 20 minutes, total: 40 minutes) did not alter systolic and diastolic blood pressure responses compared to 8 hours of prolonged sitting (300).

Simple resistance activity interruptions to sitting (squatting, calf raises; 3-minute bouts every 30 minutes, total: 27 minutes) resulted in increased resting blood flow (~43%) and shear rate (~98%) in the femoral, but not brachial artery, compared to 5 hours uninterrupted sitting in adults with overweight and obesity (279). Interruptions to sitting with calisthenic exercises (squats, arm circles, calf raises; 2-minute interruptions every 20 minutes, total: 6 minutes) increased shear rate in the brachial artery (~16%) in healthy adults (287). Performing simple leg movements such as fidgeting was also sufficient to attenuate sitting-induced decreases in popliteal artery blood flow (-19% vs -29%) and shear rate (-22% vs -43%) in healthy adults (301). Finally, brief walking interruptions to sitting (3.2 and 5.8 km/h; 2.8 and ~4.3 METs, respectively; 2-minute bouts every 20 minutes, total: 28 minutes) may also play an important role at improving concentration of hemostatic and/or pro-coagulant risk factors (e.g., fibrinogen, hematocrit, and hemoglobin) (302).

Reducing/interrupting SB vs continuous MVPA/exercise. A 30-minute bout of moderate-intensity walking (71% maximum heart rate), but not regular 2-minute bouts of moderate-or vigorous-intensity walking (53 and 79% maximum heart rate; total: 42 and 16 minutes, respectively), significantly reduced ambulatory systolic blood pressure (-3 mmHg) in adults with overweight/obesity compared to 9 hours of prolonged sitting (303).

Similarly, a 30-minute bout of moderate-intensity walking (55% heart rate reserve) reduced systolic blood pressure responses in the 4-hour period after exercise in postmenopausal females with rheumatoid arthritis (47% participants had hypertension), which was not observed following 3-minute boults of light-intensity walking every 30 minutes (24% heart rate reserve, total: 42 minutes) (213). Interestingly, resting systolic blood pressure was significantly lower in the morning after performing both a single bout and multiple 3-minute bouts of brisk walking every 30 minutes (41-42% VO 2max , total: 30 minutes) compared to a sedentary condition (-7 and -8 mmHg, respectively) in healthy male adults (304). Finally, a single 30-minute bout of moderate-intensity walking (65-75% maximum heart rate) was effective at reducing systolic and diastolic blood pressure in older adults (-3 and -1 mmHg, respectively). Performing regular 3-minute light-intensity walking interruptions every 30 minutes (3.2 km/h, 2.8 METs; total: 36 minutes) after exercising further reduced systolic blood pressure (-5 mmHg) but not diastolic blood pressure (291).

Evidence from longer-term studies

A meta-analysis has summarized data from longer-term studies investigating the effects of SB interventions conducted in free-living (-28.6 min/day of SB). Performing interruptions to sitting resulted in a small but significant reduction in systolic, but not diastolic, blood pressure (-1.1 mmHg) compared to control groups in both apparently healthy and clinical populations [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. Nonetheless, other meta-analyses of studies involving clinical population groups (134) and focused on reducing/interrupting SB with standing in adults and older adults (135) showed no change in blood pressure following intervention. A systematic review of workplace reducing/interrupting sitting interventions showed that only five out of 22 studies reported reductions in systolic blood pressure and two observed drops in diastolic blood pressure (136).

In children, an 8-month, school-based intervention using height-adjustable desks in the classroom was ineffective at reducing classroom and total daily SB (137).

Consequently, no significant changes were observed on systolic and diastolic blood pressure (137).

Regarding other hemodynamic parameters, the effect sizes for increases in shear rate in the femoral and brachial arteries following an 8-week intervention to reduce/interrupt SB at work (-38 min/8-hour workday of sedentary time and +35 min/8hour workday of standing time) were considered small (Cohen's d: 0.31 and 0.23) (305).

In a 16-week non-randomized trial, an intervention to reduce sitting (-60 min/day of sedentary, +36 min/day of standing and +30 min/day of walking time) resulted in increased femoral artery antegrade shear rate (~14%), but not in basal blood flow or retrograde shear rate, among those (≥55 years old) with increased cardiovascular risk (306).

In summary, the effects of reducing/interrupting SB on hemodynamics are inconsistent across studies and meta-analysis. It is not clear whether type, intensity, and frequency of interruptions to sitting, characteristics of population groups and/or duration of intervention affect responses to reducing/interrupting SB interventions.

Reducing/interrupting SB vs continuous MVPA/exercise. A small-scale, 12-week, multifactorial, pilot-study examined both the independent and the combined effects of exercise training (40-65% hear rate reserve, i.e., moderate to vigorous intensity) and reducing SB (replace SB with standing and LPA plus increasing daily step count by 5-10%) on blood pressure in adults with overweight/obesity (140). Reducing SB with LPA (-4.8% of daily hours in SB, +28 min/day in MVPA) resulted in significant decreases in systolic and diastolic blood pressure (-4.7 and -4.0 mmHg, respectively). Interestingly, exercise training (no change in SB + 27 min/day increase in MVPA) and exercise training combined with reducing SB (-7.3% of daily hours in SB + 45 min/day increase in MVPA) significantly reduced systolic (-7.0 and -5.9 mmHg, respectively), but not diastolic, blood pressure. There were no between-group differences with respect to improvements in systolic blood pressure (140). In contrast, 4 weeks of multiple interventions aimed at reducing SB (-53 min/day in SB with no change in MVPA) or increasing MVPA to at least 30 min day (+16 min/day of MVPA with no change in SB) had no effect on blood pressure in physically inactive adults with obesity (142).

Clinical significance

Blood pressure is one of the most commonly used hemodynamic parameters in clinical practice. There is some evidence of small improvements in systolic blood pressure (-1.1 mmHg) associated with reducing/interrupting SB [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]. Such benefits were not observed for diastolic blood pressure. It is important to highlight that mean baseline blood pressure of the participants were typically within normal ranges in available metaanalyses [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]134), which may reflect the small magnitude of reported changes following SB interventions. Importantly, these small changes in systolic blood pressure are comparable to those shown to be associated with continuous aerobic exercise and resistance exercise training in people with normal blood pressure (-0.6 to -0.8 mmHg) (307). In contrast, exercise training could be more effective at reducing diastolic blood pressure (-1.1 to -3.3 mmHg) (307). Despite the small effects of reducing/interrupting SB, even discrete reductions in blood pressure (e.g., 2 mmHg) are relevant at a population level, as they have been shown to be associated with significant reductions in the risk of all-cause, coronary diseases and stroke mortality (12,000 lives saved per year) (308).

Evidence on the effects of reducing/interrupting SB on blood pressure in individuals with hypertension is still scarce. An analysis of pooled data from 4 acute randomized crossover trials indicated that blood pressure reductions were of greater magnitude in adults with overweight/obesity and hypertension (~-10 to -13 vs -2 to -7 mmHg) following intermittent light-walking or simple resistance activity interruptions to sitting (3.2 km/h, 2.8 METs, or body-weight squatting and calf raises; 3-minute bouts every 30 minutes, total: 36 minutes) compared to those without hypertension [START_REF] Dempsey | Sitting Less and Moving More: Implications for Hypertension[END_REF].

Additionally, in a 12-month randomized controlled trial, individuals who were randomized to the group STAND+ (sit to stand desk plus goal of ≥30 minutes of additional LPA) significantly reduced sitting time by -59.2 min/8-hour workday compared to the group MOVE+ (goal of ≥30 minutes of additional LPA). These changes in physical behavior resulted in non-significant changes in cardiometabolic outcomes (234). A subgroup analysis in individuals with dysglycemia (baseline blood pressure: 135/83 mmHg) revealed larger reductions in systolic blood pressure (-6.6 mmHg) for participants randomized to STAND+ compared to MOVE+ (234). Altogether, these findings indicate that reducing/interrupting SB has the potential to improve blood pressure to a greater extent in those with hypertension.

Cardiovascular function and structure

Increasing sedentary behavior

Evidence from acute studies

Prolonged sitting may induce endothelial dysfunction (i.e., the inability of the blood vessels to dilate appropriately) and oxidative stress (309,310). Padilla and colleagues (272) observed that 3 hours of uninterrupted sitting attenuated popliteal artery shear rate (~75%) in healthy adults, but this reduction was not paralleled by a concomitant reduction in flow-mediated dilation (FMD). Conversely, Thosar and colleagues (276) reported a reduction in the superficial femoral artery FMD (~2.5 %FMD) following 3 hours of uninterrupted sitting. Restaino and colleagues (311) also demonstrated that prolonged sitting for 6 hours impairs lower-limb FMD (~5 %FMD), but not upper-limb. This is possibly because upper limb movement was not restricted in this study. These results are corroborated by meta-analytic findings showing that acute exposures to prolonged sitting result in reduced lower-limb (~2 to 5 %FMD), but not upper-limb, vascular function in healthy adults [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]120). Notably, no significant reductions were observed for exposures that were shorter than 2 hours of uninterrupted sitting [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF].

Additionally, some studies reported increases in artery stiffness, as measured by pulse wave velocity (PWV). Carotid-to-femoral PWV was increased in healthy adults following 3 hours of prolonged sitting (288, 312) and carotid-to-ankle PWV has been also shown to increase in adults with overweight/obesity and elevated blood pressure following 10 hours of prolonged sitting (313). However, these increases are rather marginal and likely not clinically relevant (314). Despite this evidence, some studies have not observed alterations in markers of macrovascular function (e.g., FMD (281,283) and PWV (281,313,315)). Some studies demonstrated that microvascular reactivity is blunted following 1.5-6-hour exposures to prolonged sitting in both upper and lower extremities, as evidenced by reductions in peak blood flow and blood flow area under the curve (AUC) (282,288,311,(316)(317)(318)(319). Studies also demonstrated impairments in tissue oxygenation index recovery rate during reactive hyperemia after ~3 hours of prolonged sitting (281,315,320). However, some studies demonstrated alteration in markers of microvascular function (e.g., shear rate (279) and blood flow area under the curve (AUC) (319)).

Seven days of lower-limb immobilization resulted in reductions in femoral artery base diameter (~5%) and vascular conductance (~23%), and increases in vascular resistance (~35%) in healthy male adults, but all parameters returned to baseline levels after resuming habitual activity for 14 days (321).

Five days of reduced step count (~12,000 to ~3,500 steps/day) reduced popliteal artery (-3 %FMD), but not brachial artery, FMD in healthy male adults (103).

Conversely, base diameter was decreased in the brachial artery (~5%, suggestive of inward vascular remodeling), but not in the popliteal artery (103). Additionally, CD31 + /CD42b -endothelial microparticles concentration (a marker of endothelial apoptosis) increased significantly by ~490% after 5 days of reduced activity (103).

Potential mechanisms. The seated posture results in greater amount of low and oscillatory shear rates in the lower extremities conduit arteries (272,276,277,288,311,316). Both oscillatory and low shear stress can increase endothelial cell-derived reactive oxygen species (ROS) and downregulate endothelial nitric oxide synthase (eNOS) expression and production of NO (a potent dilator released from endothelial cells) (322,323). It has also been proposed that increased endothelin-1 concentrations (ET-1, a potent vasoconstrictor) may upregulate ROS and reduce total plasma nitrate and nitrite, markers of NO bioavailability [START_REF] Padilla | Prolonged Sitting Leg Vasculopathy: Contributing Factors and Clinical Implications[END_REF]. Increases in ET-1 concentrations, but not NO bioavailability, have been shown following exposures to prolonged sitting (279,315). However, others did not report such increases in ET-1 concentrations (283, 312).

Evidence from longer-term studies

Unilateral leg immobilization for 12 days reduced femoral and popliteal arteries mean diameter (~7% and 14%, respectively) but did not change resting carotid artery diameter in healthy adults (295). Popliteal artery FMD increased in the immobilized leg (~7 %FMD), but not in the non-immobilized leg (295).

Clinical significance

Acute exposures to prolonged sitting resulted in significant decreases in lower-limb (-2 to -5 %FMD), but not upper-limb, in healthy adults [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]120). Similar decreases in vascular function (-3 %FMD) have been reported following 5 days of reduced step count in healthy male adults (103). FMD is a predictor of cardiovascular events in the general population (324). However, it is unclear whether these detrimental changes are likely to be sustained over time and whether upper limb FMD might also be impacted by SB.

Reducing and interrupting sedentary behavior

Evidence from acute studies

Three meta-analyses have summarized the effects of interrupting sitting on FMD [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]120,325). Two of those indicated that vascular dysfunction can be mitigated by interrupting prolonged sitting with aerobic, standing or simple resistance activities when compared to prolonged sitting (1.5 to 5 hours) (120, 325). These short-term interventions improved FMD by 1.5 to 1.9 %FMD (120,325), and increased shear rate by 12.7 S -1 in adults and older adults (325). In contrast, the most recent meta-analysis demonstrated a small, non-significant effect of PA interruptions to sitting on FMD [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF].

Of note, some original studies failed to show improvements in lower-and/or upper-limb FMD following at least one of the interruptions to SB compared to prolonged sitting [START_REF] Peddie | The Effects of Prolonged Sitting, Prolonged Standing, and Activity Breaks on Vascular Function, and Postprandial Glucose and Insulin Responses: A Randomised Crossover Trial[END_REF]279,280,283,287,326,327). Meta-regressions revealed that higher BMI is significantly associated with greater reductions in FMD responses following interruptions to sitting as compared to prolonged sitting (325), indicating that those with higher BMI may benefit more from interrupting sitting. Subgroup analysis also indicated that aerobic and simple resistance activities may be more effective than standing interruptions at improving FMD, but this comparison did not reach statistical significance due to the low number of included studies (120). Regarding frequency of interruptions to sitting, interrupting prolonged sitting with 3-minute of simple resistance activities every 30 minutes (squat, calf raises; total: 39 minutes) was more effective at increasing FMD in the femoral artery in adults and older adults with type 2 diabetes compared to a lower frequency of interruptions Change in these biomarkers, along with increased skeletal muscle activity and other systemic changes (reduced postprandial glucose responses, blood lipids, inflammatory markers, and sympathetic nervous system activity) are thought to be the potential mechanisms underpinning the effects of reducing/interrupting SB on cardiovascular function (FIGURE 5) [START_REF] Dunstan | Sit Less and Move More for Cardiovascular Health: Emerging Insights and Opportunities[END_REF]329).

- -------------------------------INSERT FIGURE 5 ABOUT HERE --------------------------------5.2.2.2. Evidence from longer-term studies Only four longer-term trials have investigated the effects of reducing/interrupting SB with standing, walking and/or other types of PA on vascular function (325). A meta-analysis summarizing the available evidence showed a significant increase in FMD (0.93 %FMD) in adults and older adults following 8 to 16 weeks of intervention (325). However, this beneficial effect was mainly driven by one study (305), with other studies suggesting no change in lower-and/or upper-limb FMD following reducing/interrupting SB (305,306,330).

Clinical significance

There is some evidence of improvements on brachial and lower-limb vascular function (0.93 %FMD) associated with reducing/interrupting SB. This change is likely to be clinically meaningful, given that a 1% increase in FMD is significantly associated with a 17% decrease in future risk of cardiovascular events (331, 332). However, the effects of reducing/interrupting SB on vascular function appear to be less pronounced relative to those observed for aerobic, resistance and combined exercise training (+2.1 to 2.8 %FMD) in adults and older adults (333). Despite this initial evidence indicating a potential benefit of reducing/interrupting SB, the evidence base is still limited, and future studies are needed to elucidate the effects of reducing/interrupting SB on vascular function and the factors that mediate intervention effects.

Cardiorespiratory fitness

Increasing sedentary behavior

Evidence from longer-term studies

Fourteen days of reduced step count (10,501 to 1,344 steps/day) resulted in a 6.6% decline in VO 2max in healthy male adults, which was significantly correlated with reductions in daily steps (108). Other studies showed similar results in healthy adults (107,112). Importantly, VO 2max returned to baseline levels by simply resuming habitual activity for 14 days (107, 112).

Clinical significance

Increasing SB results in profound decreases in VO 2max in healthy adults (e.g., -6.6% in 14 days). Changes observed over 14 days of increased SB are dramatically accelerated compared to age-related decline in VO 2max per decade (~10%, regardless of PA level) in females and males (334). Given low cardiorespiratory fitness is significantly associated with increased risk for all-cause mortality and cardiovascular events (335, 336), these findings hold important clinical implications for those who may undergo periods of reduced activity, bed rest or immobilization. In contrast, it is important to highlight these alterations can be reverted after resuming habitual daily activities (107, 112).

Reducing and interrupting sedentary behavior

Evidence from acute studies

Acutely, regular sit-to-stand transitions (stand up and return to a seated position every 1 minute for 10 minutes) significantly increased VO 2max by ~32% in healthy adults as compared to 10 minutes seated in a chair motionless (3.86 vs. 2.93 ml•kg -1 •min -1 ) (12).

Evidence from longer-term studies

A 3-year randomized controlled trial involving physically inactive and sedentary individuals with type 2 diabetes compared 1-month exercise counseling every year or standard care (233). In an ancillary analysis, when participants were divided into quartiles based on changes in PA and SB irrespective of experimental arm, the highest quartile for changes in SB (-1.53 h/day) presented a significant increase in VO 2max (4.49 ml•kg -1 •min -1 ). Importantly, increases in time spent in SB were negatively associated with changes in VO 2max , which was independent from time spent in MVPA ( 233). This finding indicates that reducing sedentary time, leading to substantial increases in total PA may be sufficient to improve cardiorespiratory fitness in a highly physically inactive and sedentary, at-risk population group.

Reducing/interrupting SB vs continuous MVPA/exercise. In a small-scale, 12week, multifactorial, pilot-study involving 57 sedentary, overweight/obese males and females, Keadle and colleagues (140) examined both the independent and the combined effects of exercise training (40-65% heart rate reserve, i.e., moderate to vigorous intensity) and reducing SB (replace SB with standing and LPA plus increasing daily step count by 5-10%) on VO 2max . Compared to control, both exercise training (no change in SB, +27 min/day in MVPA) and exercise training combined with reducing SB (-7.3% of daily hours in SB, +45 min/day in MVPA) improved VO 2max by ~10% (2.5 ml•kg -1 •min -1 and 2.9 ml•kg -1 •min -1 , respectively). Additionally, changes in VO 2max resulting from reducing SB only (-4.8% of daily hours in SB, +28 min/day in MVPA) were not statistically significant (0.2 ml•kg -1 •min -1 ) (140), indicating that improvements in VO 2max may depend on the PA intensity. Four weeks of an intervention to reduce SB (-53 min/day in SB with no change in MVPA) resulted in significant increases in VO 2max (1.9 ml•kg -1 •min -1 ) in physically inactive adults with obesity (142). This improvement in VO 2max was comparable to the effects of an intervention aiming to increase MVPA to at least 30 min day (2.2 ml•kg -1 •min -1 ; +16 min/day of MVPA with no change in SB) (142).

Clinical significance

Significant increases in VO 2max (1.9 to 4.4 ml•kg -1 •min -1 ) have been reported following interventions to reduce/interrupt SB (-1 to 1.5 h/day) (142,233). Despite these findings, evidence is still limited, and future studies are needed to elucidate the effects of reducing/interrupting SB on cardiorespiratory fitness, the clinical significance of these improvements, and the mediating factors (e.g., intensity of PA replacing SB).

MUSCULOSKELETAL SYSTEM

6.1. Skeletal muscle 6.1.1. Increasing sedentary behavior 6.1.1.1. Evidence from longer-term studies Skeletal muscle contractile activity during sitting is significantly lower than for standing and ambulatory activities [START_REF] Gao | Acute Metabolic Response, Energy Expenditure, and EMG Activity in Sitting and Standing[END_REF][START_REF] Pesola | Muscle Inactivity Is Adversely Associated with Biomarkers in Physically Active Adults[END_REF][START_REF] Tikkanen | Muscle Activity and Inactivity Periods During Normal Daily Life[END_REF]. As discussed in subtopic 4.3.1, existing studies are reasonably consistent in showing impaired anabolic responses after different periods and forms of SB. Significant losses in muscle mass have been consistently reported after only a few days of exposure to increased SB, regardless of study model.

Computed tomography scans revealed ~2% decrements in quadriceps crosssectional area (CSA) after only 5 days of a knee-immobilization protocol in healthy older males (337). After 14 days of full-leg casting, both young and older males had experienced significant decreases (~5 to 9%) in quadriceps muscle volume as assessed by magnetic resonance imaging (MRI) [START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF]. Longer time periods have also been studied (e.g., 3.5 months), and muscle fiber CSA of the vastus lateralis was shown to be dramatically reduced (-24 to -51% depending on fiber type) in older males submitted to full-leg immobilization after total knee arthroplasty, when compared to control, physically inactive individuals (338). Notably, initial quadriceps CSA was positively associated with the magnitude of muscle atrophy following 14 days of lower limb immobilization in females, but not in males (339). These findings highlight possible sex-based differences in skeletal muscle adaptations to immobilization, in which females with higher preimmobilization muscle mass are more likely to present with muscle loss.

Several studies have demonstrated that daily step reductions (~67 to 87% reduction from baseline) resulted in lean mass decrements varying between 0.5 to 4.5% in healthy male adults (107)(108)(109)235). Fourteen days of reduced daily steps (from 5,962 to 1,413 steps/day) elicited a 4% decrease in lower-limb fat-free mass in healthy older adults (110). Furthermore, using a unilateral leg model, Devries and colleagues (105) demonstrated that 14 days of step reduction resulted in an ~1.3% loss of leg fat-free mass in healthy older males. Performing concomitant unilateral resistance exercise counteracted maladaptations by increasing fat-free mass (~1.4%) in the exercised leg.

In addition, MHC distribution in the skeletal muscle shifted from slow-twitch (MHC I) towards hybrid and fast-twitch (IIa) fiber types in vastus lateralis following 20 days of reduced steps (from 14,000 to 3,000 steps/day) in healthy male adults (106), which has also been observed following periods of bed rest [START_REF] Borina | Myosin and Actin Content of Human Skeletal Muscle Fibers Following 35 Days Bed Rest[END_REF][START_REF] Gallagher | Effects of 84-Days of Bedrest and Resistance Training on Single Muscle Fibre Myosin Heavy Chain Distribution in Human Vastus Lateralis and Soleus Muscles[END_REF][START_REF] Trappe | Human Single Muscle Fibre Function with 84 Day Bed-Rest and Resistance Exercise[END_REF].

Potential mechanisms.

Bed rest studies showed that both decreased muscle protein synthesis and increased muscle protein breakdown contribute to skeletal muscle loss in healthy individuals and for those with chronic disease conditions (340,341).

Skeletal muscle homeostasis is tightly controlled by numerous anabolic and catabolic pathways, though the precise interconnection and biological actions of these actors still need to be fully elucidated (342) (FIGURE 6). In muscle atrophy conditions, several anabolic signaling pathways may be suppressed, including phosphoinositide 3-kinase (PI3K)-Akt-mechanistic target of rapamycin 1 (mTORC1), β2-adrenergic, wingless/int1frizzled (WNT/FZD), calcineurin, hippo, and/or bone morphogenetic protein (BMP). In contrast, several catabolic signaling pathways may be overactivated, including transforming growth factor-β (TGF-β), AMPK, nuclear factor kappa light chain enhancer of activated β cells (NF-κβ), glucocorticoid receptors, angiotensin, IL-6-janus kinases/signal transducers and activators of transcription (JAK/STAT), kinin, sphingolipids, notch, and/or activating transcription factor 4 (ATF4)-endoplasmic reticulum stress (342)(343)(344)(345). Finally, recent a study showed that diminished mitochondrial energetics, lipid remodeling and increased H 2 O 2 emission in hindlimb muscles of mice were early features preceding loss of muscle function ( 346). In addition, the shift in MHC distribution from slow-twitch towards hybrid and fast-twitch following bed rest [START_REF] Borina | Myosin and Actin Content of Human Skeletal Muscle Fibers Following 35 Days Bed Rest[END_REF][START_REF] Gallagher | Effects of 84-Days of Bedrest and Resistance Training on Single Muscle Fibre Myosin Heavy Chain Distribution in Human Vastus Lateralis and Soleus Muscles[END_REF][START_REF] Trappe | Human Single Muscle Fibre Function with 84 Day Bed-Rest and Resistance Exercise[END_REF] may contribute to muscle fatigue, dependance upon muscle glycogen as fuel, and reduced capacity for fat oxidation (347).

- ---------------------------------INSERT FIGURE 6 ABOUT HERE -----------------------------------6.1.1.2. Clinical significance Increasing SB results in profound decreases (1.3 to 9%) in skeletal muscle or fat-free mass in adults and older adults. Changes observed over a few days of increased SB are comparable to muscle mass decreases (~3 to 8%) over a decade after the age of 30 (348, 349). Given that low muscle mass is significantly associated with increased risk for all-cause mortality (350), these findings could hold important clinical implications for those who may undergo periods of reduced activity, bed rest or immobilization and for older adults who present higher rates of muscle mass loss compared to younger individuals (348).

Reducing and interrupting sedentary behavior

6.1.2.1. Evidence from longer-term studies Five studies investigated the longer-term effects (4 to 16 weeks) of reducing/interrupting SB on fat-free mass. Four of them showed no effect in adult office workers and adults and older adults with type 2 diabetes (351-354). In contrast, a 3-month cluster randomized trial conducted in 161 adult office workers significantly reduced sedentary time by 35 min/8-hour workday compared to control individuals (n=131), which was primarily driven by an increase in standing time. After intervention, there was an increase in total fat-free mass (0.5 kg) in the intervention group compared to the control group (355).

Overall, longer-term findings related to the effects of reducing/interrupting prolonged sitting on skeletal muscle mass have been inconsistent. The extent to which reducing/interrupting SB increases muscle mass remains unclear. Future experimental studies are warranted to investigate whether reducing/interrupting SB and which type and intensity of PA can meaningfully impact skeletal muscle mass.

Muscle strength and functioning

6.2.1. Increasing sedentary behavior 6.2.1.1. Evidence from longer-term studies Two weeks of unilateral whole leg casting significantly impaired muscle function by ~16-31% in both adults and older male adults, as measured by maximal voluntary contraction, peak torque, specific force, and rate of force development [START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF].

Interestingly, four weeks of rehabilitation (unilateral resistance training, 3 times/week) after a 2-week immobilization restored muscle function in both age groups [START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF]. Another study found that as little as 5 days of knee immobilization resulted in an 8-9% reduction in muscle strength in healthy older males (337). Four weeks of unilateral lower limb suspension (99% reduction in daily step count) reduced isometric force during plantar flexion and knee extension tasks by ~15-25% and increased isometric fluctuations for both tasks by ~12-22%. In contrast, no change was observed in EMG activity for soleus and gastrocnemius muscles (356).

Reduction in daily steps (75% reduction from baseline) for 14 days impaired muscle strength by ~8% in healthy older adults. In contrast, a rehabilitation program of the same length (i.e., 14 days) was shown to be ineffective in restoring physical function (130), indicating that this population may be at higher risk for accelerated age-related loss in muscle mass/function after transitions to reduced activity levels. Nonetheless, the literature is inconsistent, with other studies having failed to show muscle strength decrements in response to step reduction in older adults (105, 110, 164).

Clinical significance

Increasing SB results in significant decreases in skeletal muscle strength (8 to 25%) in both adults and older adults. Changes observed with increased SB are comparable to annual decreases in grip and knee extension/flexion (2.2 to 3.1% and 3.6 to 5.0%, respectively) observed in older adults (357,358). Given that low muscle strength is independently associated with increased risk for all-cause mortality regardless of muscle mass and participation in SB and leisure time PA (350), these findings could hold important clinical implications for those who may be exposed to periods of reduced activity, bed rest or immobilization, and for older adults who may be more susceptible than younger adults to muscle loss after increased SB (359).

Reducing and interrupting sedentary behavior

Evidence from longer-term studies

A previous described, a 3-year randomized controlled trial involving physically inactive and sedentary individuals with type 2 diabetes compared 1-month exercise counseling every year to standard care. Participants in the intervention group experienced a significant reduction in sedentary time (-0.9 h/day) and increase in LPA (0.8 h/day) and MVPA (8 min/day), which resulted in improved lower body strength (~19%) as compared to the control group (232). Additionally, participants were divided into quartiles based on changes in PA and SB irrespective of experimental arm. The highest quartile for changes in SB (-1.53 h/day and +10.5 min/day of MVPA) did not have different isometric muscle strength in the upper body (shoulder press) but did have significantly greater lower body strength (leg extension) as compared to other quartiles, which may have been driven by the higher participation in PA of any intensity observed in this quartile (233). Interestingly, age was negatively and positively associated with intervention-induced changes in upper and lower body strength, respectively (233).

In relation to physical functioning, a Cochrane systematic review and metaanalysis of interventions aiming at reducing SB (1 week to 1 year) in communitydwelling older adults showed low-certainty evidence related to reductions in sedentary time (-45 min/day versus control group, which was not significant) and improvements in physical function following intervention (2 studies; changes in gait speed and physical function as assessed by a physical performance battery were not significant) (360).

Another meta-analysis, including interventions aimed at increasing participation in PA (52 min/week) and reducing SB (-58 min/day) among older adults showed a small, significant increase in physical functioning (standard mean difference: 0.21) compared to the control group (361). In patients with rheumatoid arthritis, a 4-month motivational intervention aimed at reducing SB (-1.6 h/day of sedentary, 1.3 h/day of standing and +0.5 h/day of stepping time) resulted in increased physical functioning (31%) as assessed by a disease-specific questionnaire (362). Notably, this effect was sustained up to 18 months after the end of the intervention (363).

Only a few studies have investigated the effects of reducing/interrupting prolonged sitting on muscle function. Given the limited number of studies, the extent to which reducing/interrupting SB increases muscle strength and functioning and the associated clinical significance remain unclear. Experimental studies have consistently shown significant alterations in bone parameters when exposed to periods of increased SB. Lower-limb suspension has been used as a model for such investigations, and 24 days of unilateral lower-limb suspension induced losses in bone (tibia: 0.3 to 0.9%) of healthy male adults comparable to those seen after bed rest (365).

Potential mechanisms. Markers of bone resorption (urine: hydroxyproline, deoxypyridinoline, and N-telopeptide of type I collagen; serum: type I collagen carboxytelopeptide) were significantly increased during bed rest, returning to baseline levels after resuming ambulation (366).

The effects of bone loss and recovery during/following disuse are unclear in older adults and individuals with osteopenia/osteoporosis. This should be addressed by future studies, particularly given the known negative effect of aging on cellular and molecular processes throughout the different stages of bone fracture healing (367).

Clinical significance

Limb immobilization results in profound decreases in bone mineral density (~1%), primarily in weight-bearing bones in adults. These alterations are likely to be clinically significant given that the changes observed over 24 days of immobilization are comparable to annual bone loss (~1%) in older adults (357). These findings hold important clinical implications for those who may undergo bed rest and immobilization periods due to injury and for older adults, particularly postmenopausal females who present higher rates of bone loss compared to males of similar age (357).

Reducing and interrupting sedentary behavior

To our knowledge, no experimental study has investigated the effects of reducing/interrupting SB on bone metabolism, which is a promising area to be addressed in future studies. Four to six hours of exposure to prolonged, uninterrupted sitting decreased middle cerebral artery blood flow velocity and cerebrovascular conductance (~3 to 6%) in healthy adults with desk-based job as compared to baseline levels, indicating impaired dynamic cerebral autoregulation (368,369). Three hours of exposure to prolonged, uninterrupted sitting resulted in reductions in cerebrovascular conductance index (~8%) in individuals (≥55 years old) with increased cardiovascular risk (306). In contrast, 3 hours of uninterrupted sitting (with low or high mental activity) did not change cerebral blood flow in older adults, but increased blood pressure (mean arterial pressure: 8.6 mmHg) and cerebrovascular resistance (~13%), which are known to negatively impact brain health in the long-term (370). Additionally, 3 hours of prolonged, uninterrupted sitting did not affect corticospinal excitability in adult office workers (371) nor cerebral vasomotor reactivity in individuals with increased cardiovascular risk (306).

Exposure to prolonged sitting did not change plasma concentrations of brainderived neurotrophic factor (BDNF), catecholamines and related precursors or metabolites (norepinephrine, epinephrine, dopamine, DOPA, and dihydroxyphenylglycol

[DHPG]) (284). Interestingly, changes in total fatigue levels were significantly associated with increased DHPG and decreased DOPA concentrations over 4 hours of prolonged sitting, which may reflect alterations in the sympathetic nervous system in response to prolonged exposures to sitting (284).

Potential mechanisms. It has been proposed that there may be involvement of altered cerebral glucose utilization due to increased postprandial glucose responses [START_REF] Wheeler | Sedentary Behavior as a Risk Factor for Cognitive Decline? A Focus on the Influence of Glycemic Control in Brain Health[END_REF][START_REF] Chastin | How Does Light-Intensity Physical Activity Associate with Adult Cardiometabolic Health and Mortality? Systematic Review with Meta-Analysis of Experimental and Observational Studies[END_REF][START_REF] Saunders | The Acute Metabolic and Vascular Impact of Interrupting Prolonged Sitting: A Systematic Review and Meta-Analysis[END_REF][START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]; altered cortical perfusion and oxygen delivery due to alterations in cerebral (306,368,369) and peripheral vascular function [START_REF] Taylor | The Acute Effects of Prolonged Uninterrupted Sitting on Vascular Function: A Systematic Review and Meta-Analysis[END_REF]120,325), and in the supply of BDNF (372); and increased levels of inflammatory markers and reactive oxygen species (310,(373)(374)(375). Importantly, all these factors are recognized contributors to cognitive decline and dementia [START_REF] Wheeler | Sedentary Behavior as a Risk Factor for Cognitive Decline? A Focus on the Influence of Glycemic Control in Brain Health[END_REF]376,377) (FIGURE 7). With respect to glucose utilization, altered cerebral glucose utilization seems to be a response to increased circulating glucose concentration. Acute hyperglycemia leads to a reduction in regional cerebral blood flow and a spike in insulin levels to facilitate glucose clearance. Together, these two factors favor a glucose nadir. The glucose nadir can impair endocrine counter-regulation to a subsequent dip in glucose, thus exaggerating the hypoglycemic episode [START_REF] Wheeler | Sedentary Behavior as a Risk Factor for Cognitive Decline? A Focus on the Influence of Glycemic Control in Brain Health[END_REF].

SB and physical inactivity have also been shown to alter hippocampal mitochondrial and synaptic function in rats. Using selective breeding for physical inactivity, male and female sedentary Low Voluntary Runners, wild type, and High Voluntary Runner rats underwent cognitive behavioral testing, analysis of hippocampal neurogenesis and mitochondrial respiration, and molecular analysis of the dentate gyrus. Preference for physical inactivity (i.e., Low Voluntary Runners) resulted in major detriments to cognition (spatial learning and memory), brain mitochondrial respiration (coupled and uncoupled respiration), and neurogenesis (reduced AMPA receptor subunit GluA1 protein levels, which is suggested to be an initiator of Alzheimer's disease pathogenesis) compared to wild type and High Voluntary Runner rats. Of note, a significant sex-effect was noted -these differences were essentially noted in females with males being more comparable to the wild type rats (378). These findings provide evidence that selective breeding for physical inactivity has a heritable and detrimental effect on brain health and females appear to be more susceptible.

- ---------------------------------INSERT FIGURE 7 ABOUT HERE -----------------------------------Future studies are required to better understand longer-term adaptations associated to increased SB on cerebrovascular function. Mixed findings have been reported related to alterations in cerebral blood flow responses when interrupting prolonged sitting. Carter and colleagues (368) showed that 2-minute walking interruptions every 30 minutes during sitting, but not 8-minute interruptions every 120 minutes (3.6 km/h, ~2.8-3.0 METs; total: 16 minutes), increased middle cerebral artery blood flow velocity in healthy-sedentary adults when compared to prolonged sitting over 4 hours (0.6 vs -1.2 vs -3.2 cm/s, respectively) (368). In contrast, while a single 30-minute exercise bout (65-75% maximum heart rate) increased middle cerebral artery blood flow velocity (2 cm/s) in healthy older adults compared to prolonged sitting, adding frequent light-intensity walking interruptions (3.2 km/h, 2.8 METs; 3-minute bouts every 30 minutes, total: 36 minutes) following the exercise bout did not counteract the sitting-induced decrease in mean middle cerebral artery blood flow velocity over the 6.5-hour period following exercise (379). Three hours of frequent walking interruptions (self-selected pace; 2-minute bouts every 30 minutes, total: 12 minutes) during sitting with low or high mental activity (watch TV vs cognitive puzzles) did not change cerebral blood flow in older adults compared to prolonged, uninterrupted sitting (370). It also did not counteract the sitting-induced increase in cerebrovascular resistance. Frequent, 2-minute light-intensity walking interruptions every 30 minutes of sitting (self-selected pace, total: 10 minutes) prevented sitting-induced reductions in cerebral blood flow velocity and cerebrovascular conductance index in individuals (≥55 years old) with increased cardiovascular risk as compared to 3 hours of prolonged sitting (306). Other markers of cerebrovascular flow and function were not affected by interruptions to sitting (306).

Reducing and interrupting sedentary behavior

Bojsen-Møller and colleagues (371) showed that 3-minute simple resistance activities interruptions every 30 minutes of sitting (body-weight squatting, calf raises; total: ~15 minutes) increased corticospinal excitability in adult office workers compared to 3 hours of prolonged sitting, suggesting that interruptions to sitting may promote corticospinal neuroplasticity. In contrast, short interval intracortical inhibition was unchanged following interruptions to sitting (371).

Interrupting prolonged sitting with light-intensity walking (3.2 km/h, 2.8 METs; 3minute bouts every 30 minutes, total: 30 minutes) did not change plasma concentration of BDNF and, catecholamines and its precursors or metabolites as compared to 7 hours of prolonged, uninterrupted sitting in overweight and obese adults (284). In contrast, performing a single 30-minute exercise bout (65-75% maximum heart rate) with or without subsequent light-walking interruptions to sitting (3.2 km/h, 2.8 METs; 3-minute bouts every 30 minutes, total: 36 minutes) increased serum BDNF responses (by 160 and 175%, respectively) over 8 hours in older adults, relative to prolonged sitting (372).

In contrast, no differences were observed for salivary cortisol levels between sitting interrupted with a 6-minute single bout of high-intensity interval training (~84% maximum heart rate) and uninterrupted sitting in healthy male adults (380). Similarly, no differences were reported for urinary catecholamines and cortisol concentrations between six hourly 5-min bouts of moderate-walking interruptions to sitting (perceived effort: 12 -13 "somewhat hard", total: 30 minutes) and uninterrupted sitting in healthy-sedentary adults (157). It is not clear whether type, intensity, and frequency of interruptions to sitting differentially affect central and peripheral neural effects. Eight hours of prolonged, uninterrupted sitting decreased working memory, executive function, and visual learning in healthy older adults as compared to exercise followed by prolonged sitting and exercise combined with frequent light-walking interruptions to sitting (372). In contrast, 6 hours of exposure to prolonged, uninterrupted sitting did not affect cognitive function in healthy, sedentary adults (369). Other acute studies also indicate that acute exposures to prolonged, uninterrupted sitting (3 to 7 hours) do not affect cognitive function in adults and older adults (284,380,384,385). For the acute context, whilst prolonged sitting decreases cerebral blood flow (368, 369), prefrontal cortex perfusion and oxygen delivery to specific brain regions is maintained (386), which may preserve cognitive performance. Future studies are required to better understand longer-term adaptations associated to increased SB on cognitive performance.

Reducing and interrupting sedentary behavior

Evidence from acute studies

Findings on the effects of interrupting prolonged sitting on cognitive performance have been highly inconsistent. Interrupting sitting with bouts of standing, light-or moderateintensity walking improved some cognitive performance components in three studies, namely attention (387,388), executive function (387), working memory (372, 388), and psychomotor function (388) in adults and older adults. In contrast, six hourly 5-min bouts of moderate-intensity walking (perceived effort: 12 -13 "somewhat hard", total: 30 minutes) did not affect cognitive function in healthy-sedentary adults compared to prolonged sitting and a time-matched continuous bout of moderate-intensity walking (perceived effort: 12 -13 "somewhat hard") (157). No changes in cognitive performance following interruptions to sitting have been reported in other studies (157,206,284,369,380,384,385,389,390). Nonetheless, none of these studies showed detrimental effects of interruptions to sitting on cognitive performance. It is not clear whether type, intensity, and frequency of interruptions to sitting differentially affect cognitive performance. Two experimental studies reported that an acute 3-to-5-hour exposure to prolonged sitting increased plasma IL-6 (by ~38 to 50%) concentrations in healthy adults (392) and adults with central obesity (393); IL-6 is a pleiotropic cytokine with a broad range of inflammatory, immune, and hematopoietic effects. However, three other studies showed no change in this pro-inflammatory marker after 7-8 hours of uninterrupted sitting in healthy adults (369), adults with overweight or obesity (284), and postmenopausal females with rheumatoid arthritis (213). Another study reported significant decreases in high-sensitivity C-reactive protein (~91%) concentration following 6 hours of prolonged, uninterrupted sitting (369). In the adipose tissue, uninterrupted sitting increased gene expression of IL-6 and Monocyte Chemoattractant Protein-1 (MCP1), but not of Tumor Necrosis Factor-alpha (TNF-α) and .

In a two-day crossover trial, healthy male adults performed prolonged sitting (for 7 hours), standing (6 times, for a 45-minute period each time, for 7 hours) or moderateintensity exercise (60% maximum heart rate, total: 30 minutes) on day 1, and remained seated for 6 hours on day 2 (310). On day 1, plasma superoxide dismutase concentration increased by ~13% during 7 hours of prolonged sitting as compared to standing and exercise conditions, but no difference was observed for biological antioxidant potential and catalase concentrations. Postprandial concentrations of serum derivatives of reactive oxygen metabolites tended to be higher than in fasting state following 6 hours of prolonged sitting on day 2 (310).

Evidence from longer-term studies

Fourteen days of steps count reduction (81% reduction from baseline) did not alter fasting plasma TNF, IL-6, IL-15, and adiponectin concentration in healthy male adults (108). In healthy older adults, a 14-day step reduction protocol (~70 to 76% reduction from baseline) resulted in increases in plasma C-reactive protein (110,164), and 164). Surprisingly, cytokine levels remained elevated throughout a 14-day recovery period (164). In the skeletal muscle, 14 days of step count reduction (from ~9,000 to 3,000 steps/day) increased expression of key proteins in Little is known about the effects of reducing/interrupting SB on inflammatory markers and the immune system. For instance, plasma/serum IL-6 responses were not affected by frequent, short bouts of light-and moderate-intensity walking interruptions to sitting as compared to 5.5 to 8 hours of prolonged, uninterrupted sitting in adults and older adults (213,284,393). Regarding the other cytokines, frequent, 3-minute light-intensity walking interruptions to sitting (24% heart rate reserve, total: 42 minutes) decreased plasma IL-1β (~21%) and IL-10 (~17%) and increased IL-1ra (~25%) concentrations, but did not change IL-4, IL-6, IL-8, IL-17, and IFN-γ, compared to 8 hours of prolonged sitting in post-menopausal females with rheumatoid arthritis (213). These acute responses were not observed with the traditional single 30-minute bout of moderateintensity walking (55% heart rate reserve) performed early in the morning (213). In a crossover trial, performing multiple standing bouts (6 times, total: 4.5 hours) or a moderate-intensity exercise bout (60% maximum heart rate, total: 30 minutes) protected against the increase in plasma superoxide dismutase observed following 7 hours of prolonged sitting in healthy male adults, with no differences between standing interruptions and continuous exercise trials (310). It is not clear whether type, intensity, and frequency of interruptions to sitting differentially affect markers of inflammation and oxidative stress.

Regular light-intensity and simple resistance activity interruptions to sitting over 7 hours (3.2 km/h, 2.8 METs or body-weight squatting, calf raises; 3-minute bouts every 30 minutes, total: 36 minutes) increased plasma lysoalkylphosphatidylcholine (associated with anti-inflammatory pathways) and alkenylphosphatidylcholine (associated with antioxidant capacity), and decreased diacylglycerols and triacylglycerols (associated with pro-inflammatory pathways) concentrations, but did not affect other lipid species and subspecies, in adults and older adults with overweight/obesity and type 2 diabetes (396). In contrast, a 30-minute bout of moderate-intensity walking (55% heart rate reserve), but not light-intensity walking interruptions to sitting (24% heart rate reserve, 3-minute bouts every 30 minutes, total: 42 minutes), modified serum concentration of 6 lipid classes and subclasses in a direction that indicates reduction in inflammation and platelet activation, and increase in antioxidant capacity in post-menopausal females with rheumatoid arthritis (213).

Moderate-intensity walking interruptions to sitting over 5 hours (5.8 km/h, ~4.3 METs; 2-minute bouts every 20 minutes, total: 28 minutes) resulted in increased

Clinical significance

Currently, evidence related to the effects of reducing/interrupting sitting is restricted to acute exposures. Although small improvements were shown in some inflammatory markers, it remains unclear whether these changes are clinically meaningful and sustained over time. Longer-term investigation into the effects of reducing/interrupting SB on inflammatory responses is warranted, particularly in population groups characterized by a low-grade inflammatory profile and/or high-grade systemic inflammation.

SUMMARY OF PHYSIOLOGICAL IMPACTS AND FUTURE DIRECTIONS

The rapid accumulation of epidemiological and experimental evidence on SB over the past 20 years has provided a foundation for understanding the physiology of SB. To date, evidence on the physiological effects of exposures to increased SB and the potential impact of reducing and interrupting SB raise several pertinent questions, research needs and opportunities. These include: 1) how evidence on physiological consequences of SB relates to the already vast knowledge base on physical inactivity (lack of sufficient exercise); 2) what are the effects of reducing/interrupting SB on acute/chronic physiological processes or health outcomes and the specific mechanisms involved; and 3) how the evolving knowledge about reducing/interrupting SB can provide rational mechanistic bases for interventions and future clinical and public health recommendations. Hereafter, we provide a summary of available evidence and a perspective on some of the priority areas for future work in SB physiology.

What are the effects of sedentary behaviors on physiological systems?

From a physiological perspective, the evidence to date indicates the numerous physiological responses resulting from increasing SB (TABLE 3). To summarize, excessive and prolonged SB leads to insulin resistance, vascular dysfunction, shift in substrate use towards carbohydrate oxidation, shift in muscle fiber from oxidative to glycolytic type, reduced cardiorespiratory fitness, loss of muscle mass and strength, and bone mass, and increased total body fat mass and visceral fat depot, blood lipid concentrations, and inflammation. These adaptations relate to those reported for physical inactivity (30-33, 397, 398).

- -------------------------------INSERT TABLE 3 ABOUT HERE --------------------------------A standing question is whether SB (too much sitting) and physical inactivity (too little exercise) lead to similar or distinct adaptations in relation to the number of physiological systems being impaired, magnitude of changes, and mechanisms. This question is challenging to address with the current available study models. However, bed rest studies that tested the effects of exercise training to prevent the physiological adaptations to bed rest can provide some insights. While individuals in strict bed rest are deficient in both LPA and MVPA, those who are concomitantly subjected to bed rest and exercise training are only deficient in LPA. Therefore, bed rest combined with exercise represents a model to study the adaptations and potential mechanisms of exposures to a highly sedentary yet physically active condition. Findings from these studies suggest excessive participation in SB results in adverse effects, at least for some outcomes, even in the presence of large volumes of aerobic and/or resistance exercise, i.e., above current guidelines (34) (TABLE 4). This supports that SB and physical inactivity likely coexist in a continuum related to energy demand and muscle contraction, and that the magnitude of maladaptations following physical inactivity seems to be more pronounced than those of SB in most physiological systems [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF], but it is not clear whether these behaviors differentially affect physiological systems or present distinct underpinning mechanisms. It also highlights the potential benefits of regular non-exercise activity and/or muscle contractions.

- -------------------------------INSERT TABLE 4 ABOUT HERE --------------------------------Our premise in this review is that the adaptations to increasing SB are not the opposite of adaptations to reducing/interrupting SB (i.e., increasing PA). Accordingly, evidence from each paradigm was addressed in separate sections. Notably, a few studies in rodents and humans have shed some light on potential distinct adaptations to increasing SB vs increasing non-exercise activity (e.g. LPA) vs MVPA/exercise training.

For example, alterations in heparin-released and intracellular LPL activity decreased mono-exponentially in both type 1 and predominantly type 2 muscles after 12 hours of limb unloading. These alterations were rapidly reversed with light-intensity contractile activity in both soleus and quadriceps muscles [START_REF] Hamilton | Role of Low Energy Expenditure and Sitting in Obesity, Metabolic Syndrome, Type 2 Diabetes, and Cardiovascular Disease[END_REF][START_REF] Bey | Suppression of Skeletal Muscle Lipoprotein Lipase Activity During Physical Activity: A Molecular Reason to Maintain Daily Low-Intensity Activity[END_REF][START_REF] Bey | Patterns of Global Gene Expression in Rat Skeletal Muscle During Unloading and Low-Intensity Ambulatory Activity[END_REF], but MVPA/exercise training did not enhance LPL regulation in type 1 muscles and type 2 muscles that were not recruited during running [START_REF] Hamilton | Exercise Physiology Versus Inactivity Physiology: An Essential Concept for Understanding Lipoprotein Lipase Regulation[END_REF][START_REF] Hamilton | Role of Local Contractile Activity and Muscle Fiber Type on LPL Regulation During Exercise[END_REF]. In addition, LPP1/PAP2A has been proposed as a potential gene that modulates maladaptation related to increased SB/inactivity in both humans and rats, but exercise has been found to be ineffective at counteracting alterations in LPP1/PAP2A in both species [START_REF] Miokovic | Differential Atrophy of the Postero-Lateral Hip Musculature During Prolonged Bedrest and the Influence of Exercise Countermeasures[END_REF]. Finally, a clinical study compared the effects of 2 months of exercise training in sedentary male adults with the effects of 1 month of reducing non-exercise PA (i.e., increased SB) in active male adults. Notably, the deleterious effect of increased SB was more marked than the beneficial effects of exercise training following current PA guidelines for dietary fat oxidation, fasting and postprandial insulin concentration/response, postprandial triglycerides response, and fat-free and fat mass (127). In contrast, the magnitude of changes in VO 2max was more pronounced following exercise training than increased SB (127). Altogether, these findings suggest distinct mechanisms may underpin adaptations to increased SB and exercise training. Further investigations are however clearly required.

It is critical that well-powered, rigorous studies are conducted to examine the acute and chronic physiological adaptations to imposed SB. Studies aiming to investigate the physiological consequences of imposed SB ('problem-focused' approach) should focus on recruiting individuals with low sedentary time who will experimentally modify their usual activity (low SB and high LPA) to more sedentary states (higher SB and lower PA of any intensity). This concept is analogous to the paradigm put forward initially by Booth and Lees (399) stating that appropriate models of physical inactivity should utilize an approach whereby physically active individuals become physically inactive (400). To effectively isolate the effects of physical inactivity from those of SB, studies should focus on recruiting individuals who do not meet current PA guidelines (150 min/week of MVPA) or perform regular exercise training. Though bed rest can be a well-controlled and useful model to study SB physiology, studies should also focus on increased SB with behaviors more commonly seen in free-living (i.e., sitting not strict lying down).

Future studies should (i) investigate whether differential changes in muscle mass and fiber type are associated with SB-related metabolic adaptations; (ii) determine the molecular and cellular mechanisms underpinning adaptations to increasing SB, e.g., using 'omics' approaches (genomics, epigenomics, transcriptomics, proteomics, metabolomics, etc.); (iii) implement different volumes and types of PA/exercise to determine how much PA and/or exercise is needed to offset the adverse effects of increased SB; (iv) include rigorous measurements and control PA level at baseline and during the study protocol, preferably via device-based measures, which is a major limitation of free-living studies to increase SB (i.e., reduced step count and immobilization). Given most of available evidence is limited to healthy male adults, future studies should focus on investigating the effects of increasing SB in females, children, adolescents, older adults, more-diverse population groups and individuals at risk for or with chronic conditions. This will assist in providing the strong rational biological bases that are much needed for improving our understanding of the physiology of SB and its multiple health consequences.

What are the beneficial effects of reducing/interrupting sedentary behavior?

Acutely, reducing/interrupting SB improves postprandial glucose and insulin responses, systolic and diastolic blood pressure, and lower-limb vascular function in adults and older adults (TABLE 5) (75-77, 119, 120, 325). In the longer term, reducing/interrupting SB interventions result in small improvements on body weight (-0.6 kg), waist circumference (-0.7 to -1.5 cm), percent body fat (-0.3 to -0.7%), fasting glucose (-2.5 mg/dL), insulin (1.4 pM), HbA1c (-0.2%) and HDL (0.04 mM) concentrations, systolic blood pressure (-1.1 mmHg), and brachial and lower-limb vascular function (0.93 %FMD) in adults and older adults (TABLE 5) [START_REF] Hadgraft | Effects of Sedentary Behaviour Interventions on Biomarkers of Cardiometabolic Risk in Adults: Systematic Review with Meta-Analyses[END_REF]134,135,325). There is more-limited evidence for other health outcomes and physiological systems. Despite this initial evidence, findings from acute and longer-term studies aimed at reducing/interrupting SB are inconsistent with other findings of no benefit for some outcomes. Overall, effects of reducing/interrupting SB are small, and likely not to be clinically and physiologically meaningful in healthy population groups, but plausibly, the effects are likely to be larger in less-healthy populations. Future studies need to investigate whether these small improvements on health outcomes observed with reducing/interrupting sedentary behavior associate with reduced risk of chronic diseases and early mortality.

- -------------------------------INSERT TABLE 5 ABOUT HERE --------------------------------For studies focused on the consequences of reducing/interrupting SB ('solution focused' treatment paradigm), the emphasis should be directed towards recruiting physically inactive-sedentary individuals with modification of their typical low PA level (high SB and low PA) to higher PA levels (low SB and high PA of any intensity). This Future studies should focus on reporting data by key factors, including, but not limited to, age groups (preferably by smaller age groups, such as 5-year increments), BMI, sex/gender, race/ethnicity, genetic profiles, menopausal and pregnancy status, medications, dietary habits, cardiorespiratory fitness, baseline SB/PA/exercise levels, sleep duration and quality, and populations at increased risk of or with chronic diseases, as indicated by the 2020 World Health Organization Guidelines Development Group (401,402). Identifying whether such factors hold significant importance that will help identify more 'at risk' population groups and those who may derive more benefits from reducing/interrupting SB. Harmonized analysis using individual participant data and dose-escalation trials could also be implemented to evaluate the effect of different "doses" of SB reductions on physiological outcomes. More robust evidence on both the direct and indirect underlying cellular and molecular mechanisms associated with reducing/interrupting SB is also needed. This may be garnered through the collection of tissue samples (e.g., muscle, bone, adipose tissue), including more direct and integrated physiological measurements, rather than surrogate markers. This will assist in providing the strong rational biological bases that are much needed for improving our understanding of the physiology of reducing/interrupting SB, and refining intervention strategies and guidelines to address SB as a clinical and public health problem.

What is the 'optimum' Frequency, Intensity, Time and Type of activities when reducing/interrupting sedentary behavior?

Elements pertaining to SB FITT may influence the effects of reducing/interrupting SB on glucose responses, which is the most-studied outcome. To summarize, it seems that more frequent (2-6-minute bouts every 20-60 minutes) and higher-intensity (light-to moderate-intensity) interruptions yields more pronounced improvements in relation to postprandial glucose responses than less frequent (every 120 minutes or more) and lower-intensity (i.e., standing still) interruptions to sitting (75-77, 181, 198, 206). Despite being beneficial, it is still unclear whether there are differences between higher frequencies of interruptions (e.g., every 20 minutes vs 30 minutes) or between intensities (i.e., light vs moderate vs vigorous). There are also some inconsistencies across original studies and meta-analysis on what is the ideal SB FITT. There is morelimited evidence on the influence of FITT elements of an intervention on changes in health outcomes other than glycemia.

To summarize, current evidence does not allow us to conclude what is the desirable FITT to reducing/interrupting SB and significantly affect physiological systems in healthy adults and older adults, and those at-risk or with chronic diseases. The "ideal" or "optimal" FITT of SB and PA elements is likely to be based on the requirements, context, and activity/health status of the subpopulation, rather than a "one size fits all" approach. However, in terms of potential countermeasures applicable to the population, it may be that certain minimal combinations or criteria of mode or posture (e.g., active sitting, fidgeting, acute or extended postural changes, standing, activities involving resistance, and/or sit-to-stand transitions), volume or intensity (e.g., LPA or MVPA), or patterning (e.g., activity bout, active around meals, or standing length/accumulation) of physical movement are all that is required to derive physiological benefit.

Another pertinent question is whether regular interruptions to sitting would be more beneficial than performing the traditional continuous bout of activity. Acutely, reductions in postprandial glucose responses are more pronounced following frequent interruptions to sitting compared to a time-matched continuous bout of activity [START_REF] Loh | Effects of Interrupting Prolonged Sitting with Physical Activity Breaks on Blood Glucose, Insulin and Triacylglycerol Measures: A Systematic Review and Meta-Analysis[END_REF]197,198,213). In contrast, a continuous bout of PA in the morning is more effective at attenuating postprandial triglycerides responses and systolic blood pressure than frequent interruptions to sitting (213,214,257,258,303). Nonetheless, evidence on the differential effects of frequent interruptions to sitting versus a continuous bout of PA/exercise is still limited to acute settings, a select number of health outcomes, and generally healthy population groups. Evidence on the combined effects of exercise and reducing/ interrupting SB is also very limited. This is a critical gap in the literature, given the combination of both strategies is what has been currently recommended in public health guidelines for all age and population groups.

Future studies should address the evidence gap on the 'optimum' FITT of activities when reducing/interrupting SB, particularly in the longer term. To do so, studies should include experimental groups with different FITT of interruptions to SB versus a sedentary (control) group. If possible, groups should be matched for energy expenditure and FITT elements other than the one being tested should remain unchanged. For example, when testing the influence of frequency, total duration, intensity, and type of activity should be the same across experimental groups. Studies should also (i) include detailed description on how participants were instructed to reduce/interrupt SB (as per all FITT elements); (ii) provide participants with specific and measurable goals, so that adherence to FITT prescription can be objectively assessed;

(iii) describe how adherence was assessed and check adherence to the prescribed intervention throughout the study protocol; (iv) report behavioral outcomes that reflect changes in the FITT element being tested, e.g., report changes in the number of daily interruptions to SB and number/duration of prolonged SB bouts when testing the effects of different frequencies of interruptions to SB; and (v) report adherence to the prescribed intervention (as per FITT elements being measured/controlled) using objective assessment (e.g., accelerometers, HR monitors, wearables, etc.) (TABLE 2).

This will improve the study design, data analysis, reporting/data harmonization in future studies, and hence the robustness of the findings. It will ultimately assist with refining preventative strategies and guidelines to combat excessive SB.

CONCLUSIONS

116 SB is highly prevalent in daily living and most of the population is exposed, to a greater or lesser extent, to the health risks of too much sitting. Excessive SB negatively impacts a multitude of physiological systems, leading to insulin resistance, vascular dysfunction, shift in substrate use towards carbohydrate oxidation, shift in muscle fiber from oxidative to glycolytic type, reduced cardiorespiratory fitness, loss of muscle mass and strength, and bone mass, and increased total body fat mass and visceral fat depot, blood lipid concentrations, and inflammation. From a physiological perspective, exposures to increased SB result in maladaptations that are similar to those that have been reported for physical inactivity, but generally lower in terms of magnitude.

Sedentary behavior Any waking behavior characterized by a low EE (≤1.5

METs) while in a sitting, reclined or lying down posture.

(1, 403)

Pattern

The manner in which sedentary behavior is accumulated, for example, timing of the day, duration and frequency of bouts and breaks.

(1)

Standing

The act of one maintaining an upright position while supported by one's feet.

( ( [START_REF] Bull | World Health Organization 2020 Guidelines on Physical Activity and Sedentary Behaviour[END_REF][START_REF] Piercy | The Physical Activity Guidelines for Americans[END_REF] Legend: EE, energy expenditure; HR, heart rate; %HRR, percentage of heart rate reserve; METs, metabolic equivalent.

Body mass and composition

Body weight (kg) NA --0.6 (-0.9 to -0. behavior reduce blood flow and shear stress, increasing endothelin-1 and attenuating nitric oxide, subsequently leading to reduced flow-mediated dilation, particularly in the lower limbs, and increased blood pressure. In addition, muscle inactivity, insulin resistance, hyperlipemia, inflammation, oxidative stress, and increased sympathetic nervous system activity may further augment the consequences of sedentary behavior on vascular health. B) Reducing/interrupting sedentary behavior may be a potential strategy to improve cardiovascular health by attenuating sedentary behavior-induced maladaptations in cardiovascular and metabolic health [START_REF] Dunstan | Sit Less and Move More for Cardiovascular Health: Emerging Insights and Opportunities[END_REF]329). Abbreviations: ET1, endothelin-1; NO, nitric oxide. Reducing/interrupting sedentary behaviors may be a potential strategy to attenuate sedentary behavior-induced maladaptations in the central nervous system function [START_REF] Wheeler | Sedentary Behavior as a Risk Factor for Cognitive Decline? A Focus on the Influence of Glycemic Control in Brain Health[END_REF].

Abbreviation: BDNF, brain-derived neurotrophic factor.

1 .

 1 INTRODUCTION ................................................................................................. 6 2. AN OPERATIONAL FRAMEWORK FOR SEDENTARY BEHAVIOR PHYSIOLOGY RESEARCH ............................................................................................................... 7 3. BODY WEIGHT AND ENERGY BALANCE ....................................................... 26 4. INTERMEDIARY METABOLISM ....................................................................... 36 5. CARDIOVASCULAR AND RESPIRATORY SYSTEMS .................................... 66 6. MUSCULOSKELETAL SYSTEM ....................................................................... 84 7. CENTRAL NERVOUS SYSTEM ........................................................................ 93 8. IMMUNE SYSTEM .......................................................................................... 101 9. SUMMARY OF PHYSIOLOGICAL IMPACTS AND FUTURE DIRECTIONS .. 106 10. CONCLUSIONS .............................................................................................. 115 REFERENCES ....................................................................................................... 118 systolic blood pressure, and vascular function. Evidence is more limited for other health outcomes and physiological systems.

1 .

 1 Increasing sedentary behavior 3.1.1.1. Evidence from longer-term studies

3. 1

 1 .1.2. Clinical significance Increases in body fat mass induced by models involving increased SB are likely clinically relevant. Specifically, increases in body fat mass reported in reduced step studies (3 to 14%) are considerably more pronounced than longitudinal changes observed in the general population. A populational cohort study showed an ~1% (0.7 kg) increase in body fat mass over the course of 12 years (131). Of concern, measures of adiposity (BMI, visceral fat mass or central adiposity and body fat percentage) are positively associated with increased risk of all-cause, cardiovascular disease and cancer mortality (132), and 21 major chronic diseases (133).

  ) and the largest lean tissue mass in adults without obesity (222). Lack of muscle contractions have been one of the proposed mechanisms for SB-related impairments in glucose metabolism. During periods of SB, skeletal muscle accounts for only 15% of whole-body glucose (223), whereas it accounts for more than 80% of the insulinstimulated glucose disposal and is quantitatively the most dominant tissue during exercise (221). Hamilton and colleagues (224) developed a physiological method of muscle contractile activity to magnify and sustain soleus oxidative metabolism (~88% type I slow-twitch fibers) through performing 'soleus push ups' (~1.3 and 1.7 METs, 50 to 100 contractions/min). Sustained continuous soleus contractile activity improved systemic metabolic regulation, by reducing 3-hour post-load glucose and insulin responses (-39-52% and -41-60%, respectively) and 2-hour post-load glucose concentration (-29-46 mg/dL) in adults and older adults (BMI: 20-43 kg/m 2 ). Sustained contractions also increased energy demand (91 kcal/h above sedentary control) and local carbohydrate oxidation (100-200 mg/min above sedentary control) (224). These findings indicate that increasing local contractile activity in small oxidative muscles can be a potent strategy for improving systemic metabolic regulation.In the skeletal muscle, 5 hours of frequent, walking interruptions during sitting (3.2 km/h, 2.8 METs, 2-minute bouts every 20 minutes, total: 28 minutes) altered expression of 10 genes involved in carbohydrate metabolism, including increased gene expression of dynein light chain (DYNLL1), which may regulate translocation of the GLUT-4, and pyruvate dehydrogenase kinase 4 (PDK-4), which inhibits the pyruvate dehydrogenase complex and increases glucose utilization in adults with overweight/obesity (225). Five hours of interrupting prolonged sitting with light-and moderate-intensity walking(3.2 km/h, 2.8 METs and 5.8 km/h, ~4.3 METs, respectively;

  analyses. It is not clear whether type, intensity, and frequency of interruptions to sitting, characteristics of population groups and/or duration of intervention affect responses to reducing/interrupting SB interventions.Reducing/interrupting SB vs continuous MVPA/exercise. A small-scale, 12-week, multifactorial, pilot-study examined both the independent and combined effects of exercise training (40-65% hear rate reserve, i.e., moderate to vigorous intensity) and reducing SB (replace SB with standing and LPA plus increasing daily step count by 5-10%) on markers of insulin action in adults with overweight/obesity (140). Exercise training combined with reducing SB (-7.3% of daily hours in SB and + 45 min/day increase in MVPA) was the most effective intervention to improve markers of insulin action. This approach improved insulin sensitivity (17.8%), as assessed by a composite insulin-sensitivity index, and decreased 2-hour insulin concentration (-30.5 uIU•mL -1 ) and post-load glucose responses (-19.4%). Reducing SB alone (-4.8% of daily hours in SB, +28 min/day in MVPA) reduced fasting insulin concentrations (-3.6 uIU•mL -1 ) but, surprisingly, concomitantly increased 2-hour glucose (+26.5 mg/dL). In contrast, exercise training alone (no change in SB + 27 min/day increase in MVPA) did not improve any marker of insulin action. In addition, exercise and SB interventions, when performed independently, did not affect fasting and post-load glycemia (140). Similarly, 4 weeks of interventions aiming at reducing SB (-53 min/day in SB with no change in MVPA) or increasing MVPA to at least 30 min/day (+16 min/day of MVPA with no change in SB) did not change fasting glucose and HbA1c concentrations in physically inactive adults with obesity (142).

  . The changes in the oxidative rate of dietary saturated fatty acid have been associated with lower clearance and uptake by peripheral tissues and decreases in the gene expression of CD36, fatty acid binding protein [FABPpm], CPT1 and acyl-CoA synthetase long chain family member 1 [ACSL1]. Despite a reduction in adipose tissue lipolysis (245, 246), excess of plasma lipids has been shown to enhance fat accumulation in the visceral adipose depot (247) and ectopic fat storage in muscle, liver and bone (170, 248-250). Fat accumulation is known to impair the function of the tissues. In this line, a recent study in non-human primates (Macaques)

  , 3-hydroxybutyrate, apolipoproteins C-II and C-III (257), apolipoproteins B-48 and B-100, total ketone bodies, and acetoacetic acid (215) following interruptions to sitting (standing and moderate-intensity walking) in healthy adults, adults with overweight/obesity and postmenopausal females.

  effects of SB interventions (range: 6 to 24 weeks) showed that lipid profile was not ameliorated despite significant decreases in total sedentary time (-64 min/day) and increases in walking time(27 min/day) in individuals with overweight, obesity, type 2 diabetes, cardiovascular, neurological/cognitive, or musculoskeletal diseases (134). A meta-analysis of studies aiming at replacing SB with standing time (mean follow-up: 3.8 months) showed that significant increases in total standing time(1.3 h/day) in adults had no effect on lipid profile (135). Another systematic review of studies also concluded that SB interventions did not improve blood lipids in most published studies (13 out of 18 studies) (136). Furthermore, skeletal muscle lipidome was largely unaffected after three weeks of an intervention consisting of frequent active interruptions to sitting (increase of 744 steps/day from baseline, no change in SB) in adults with central obesity(229).Taken these data together, it is evident that results on the effects of reducing/interrupting SB on markers of lipid metabolism are inconsistent across studies and meta-analysis. It is not clear whether type, intensity, and frequency of interruptions to sitting, characteristics of population groups and/or duration of intervention influence the responses to reducing/interrupting SB interventions. Reducing/interrupting SB vs continuous MVPA/exercise. A small-scale, 12-week, multifactorial, pilot-study examined both the independent and the combined effects of exercise training (40-65% hear rate reserve, i.e., moderate to vigorous intensity) and reducing SB (replace SB with standing and LPA plus increasing daily step count by 5-10%) on blood lipids in adults with overweight/obesity (140). Reducing SB with nonexercise PA (-4.8% of daily hours in SB, +28 min/day in MVPA) and exercise training combined with reducing SB (-7.3% of daily hours in SB + 45 min/day increase in MVPA) did not change fasting total cholesterol, HDL, and triglycerides concentrations. In contrast, exercise training only (no change in SB, + 27 min/day increase in MVPA) significantly reduced triglyceride concentrations (-0.4 mmol•L -1 ) (140). Four weeks of intervention aiming at reducing SB (-53 min/day in SB with no change in MVPA) or increasing MVPA to at least 30 min/day (+16 min/day of MVPA with no change in SB) did not improve HDL, LDL and triglyceride concentrations in physically inactive adults with obesity (142).

1 .

 1 Evidence from longer-term studies Most evidence related to the effects of changing sedentary and physically-active behaviors on protein metabolism has focused on skeletal muscle. In order to further understand how sedentary-induced hypokinesia may affect protein metabolism in skeletal muscle, it is important to acknowledge that muscle mass is regulated by an intricate and coordinated balance between daily fluctuations in muscle protein synthesis and breakdown (i.e., muscle protein balance) (261, 262). While breakdown is considered somewhat stable in non-pathological conditions, muscle protein synthesis is modulated by anabolic stimuli such asPA and nutrition (261, 262). This indicates that SB-related mechanical unloading of muscles may lead to muscle wasting(263, 264).

  (i.e., squatting, calf raises; 6-minute bouts every 60 minutes, total: 36 minutes), for which FMD did not change (283). It is not clear whether intensity of interruptions differentially affects FMD responses. Reducing/interrupting SB vs continuous MVPA/exercise. Continuous exercise (60 min/day of MVPA), but not substituting sitting with 5-6 h/day of LPA, over 4 days improved circulating markers of endothelial disfunction in adults with normal weight, overweight and type 2 diabetes (328). Potential mechanisms. It has been suggested that changes in vascular function may be mediated by decreases in plasma ET-1 concentration (279, 315) and increases in plasma nitrate/nitrite concentration and NO bioavailability/endothelin-1 ratio (315).

1 .

 1 Evidence from longer-term studies Increased resorption and decreased formation are considered to be the primary drivers of immobilization-induced bone loss in weight-bearing bones (364).

7. 1 . 2 . 2 .

 122 Evidence from longer-term studies The effect size for decreasing sedentary time can be considered large following an 8week e-health intervention to reduce/interrupt SB at the workplace versus a control condition (-38 min/8-hour workday of sedentary time and +35 min/8-hour workday of standing time) (305). As for changes in cerebrovascular function, large effects were observed for the change in gain (Cohen's d: 1.25) and normalized gain (Cohen's d: 0.91). Effect sizes were considered small to moderate for all other outcomes (Cohen's d: 0.00 to 0.74), indicating little to no improvements in cerebrovascular function following intervention among healthy adult office workers (305). In a 16-week nonrandomized trial, an intervention to reduce sitting (-60 min/day of sedentary, +36 min/day of standing and +30 min/day of walking time) resulted in increased resting cerebral blood flow velocity (~6%) and cerebrovascular conductance index (4%) among individuals (≥55 years old) with increased cardiovascular risk (306); no alterations in cerebral autoregulation, cerebral vasomotor reactivity and cardiac baroreflex sensitivity were observed. These findings indicate an overall increase in cerebral perfusion after SB reduction intervention.7.1.2.3. Clinical significanceSmall increases in cerebral blood flow, but not cerebrovascular function, have been reported following 16 weeks of reducing/interrupting SB (306). It is important to highlight that these improvements are in line with those observed following exercise programs in older adults(381, 382). It should be noted that even conditions such as Alzheimer's disease do not markedly affect cerebrovascular function (383). This highlights the robustness of the cerebrovascular system to regulate fluctuations in cerebral blood flow and may justify the small magnitude of changes observed following PA/SB interventions. Given the limited number of studies, the extent to which reducing/interrupting SB alters cerebrovascular flow and function, as well as the clinical significance of these findings remain imprecise. Future studies are required to better understand adaptations associated to reducing/interrupting SB in the central nervous system.

7. 2 . 2 . 2 . 1 .

 2221 Evidence from longer-term studies An 8-week e-health intervention to reduce/interrupt SB within the workplace resulted in large reductions in sedentary time (-38 min/8-hour workday of sedentary time and +35 min/8-hour workday of standing time). Effect sizes were considered small for changes in work productivity (Cohen's d: 0.47) and concentration/focus (Cohen's d: 0.00), indicating little to no improvements following intervention among healthy adult office workers (305). In adults (≥50 years old) with knee osteoarthritis, an intervention aimed at increasing MVPA and reducing SB did not significantly increase working memory and episodic memory (391). Changes in MVPA and SB were not associated with changes in cognitive function during the study protocol (391). 7.2.2.3. Clinical significance Acute and longer-term findings related to the effects of interrupting prolonged sitting on cognitive performance have been highly inconsistent. It remains unclear the extent to which reducing/interrupting SB alters cognitive function, but studies indicate that this strategy does not negatively affect cognition. Longer-term investigation into the effects of reducing/interrupting SB on cognitive performance is warranted. Immunity and inflammatory responses 8.1.1. Increasing sedentary behavior 8.1.1.1. Evidence from acute and multi-day studies

  inflammatory signaling pathways (i.e., c-Jun N-terminal kinase [JNK], NF-kβ inhibitor-α [Ikβα] and toll-like receptor 4 [TLR4]) (130) and macrophages infiltration in healthy older adults, likely due to an increase in transient muscle edema and/or minor myofiber damage (165). 8.1.1.3. Clinical significance Exposures to prolonged SB increased some inflammatory markers, including plasma Creactive protein, IL-6 and TNF-α concentrations, in both adults and older adults. It is unclear whether these detrimental changes are clinically meaningful and sustained over time. Given that low-grade inflammation is important in the pathogenesis of cardiovascular and other chronic diseases (394, 395), longer-term studies should further investigate whether the altered inflammatory responses following increased SB are associated with alterations in other physiological systems. 8.1.2. Reducing and interrupting sedentary behavior 8.1.2.1. Evidence from acute and multi-day studies

  Nicotamide N-methyltransferase (NNMT; modulates anti-inflammatory and antioxidative pathways) gene expression in the skeletal muscle as compared to prolonged, uninterrupted sitting in adults with overweight/obesity (225). In subcutaneous abdominal adipose tissue, interrupting sitting with light-intensity walking hours (3.2 km/h, 2.8 METs; 2-minute bouts every 20 minutes, total: 28 minutes) resulted in upregulation of immune function and downregulation of inflammatory pathways (total of 8 pathways) as compared to uninterrupted sitting in adults with overweight/obesity (228).

  includes conducting randomized controlled trials (with appropriate control groups) of longer duration (>12 weeks), familiarizing/habituating study participants to interventions and study procedures, continuously monitoring the adherence to interventions, controlling for potential confounders during the design, conduct and analysis of the trial, and analyzing experimental data considering both the intention to treat principle and adherence to the interventions.Studies should include rigorous measurements of primary, secondary, and exploratory outcomes, and report all possible outcomes and indexes being measured (e.g., acute studies measuring glucose concentrations should report not only AUC, but also mean glucose concentration during the protocol). The monitoring of PA level at baseline, preferably via device-based measures, to access eligibility and during study follow-up can enhance scientific rigor. Longer-term, randomized controlled trials should also combine device-based measures with self-reported (e.g., questionnaires and diaries) to gather information on activity type and context of SB and PA (299).

  Longer-term interventions aimed at reducing/interrupting SB have only resulted in small improvements on body weight, waist circumference, percent body fat, fasting glucose, insulin, HbA1c and HDL concentrations, systolic blood pressure, and vascular function in adults and older adults. Because of inconsistencies in the reported effects of reducing/interrupting SB, the clinical significance of these findings still remains somewhat unclear. Although the 'sit less, move more and exercise' message currently promoted by contemporary public health guidelines has received a clear general consensus based on a growing body of epidemiological findings, further experimental studies are needed to elucidate the physiological effects of interventions combining exercise and reduction/interruptions to sitting. Nonetheless, reducing/interrupting SB is a low-risk strategy and is likely relevant from a population point of view, particularly given that it can serve as a stepping stone to increase participation in PA/exercise for those who do not, or have significant challenges to, achieve the minimum guidelines on MVPA. 122. Duvivier B, Schaper NC, Koster A, van Kan L, Peters HPF, Adam JJ, Giesbrecht

  ) Passive Any waking activity characterized by an EE ≤ 2.0 METs while standing without ambulation. produced by the skeletal muscles that result in an increase in EE above resting levels. (404) Exercise Refers to a physical activity that is planned, structured, repetitive, and purposeful (i.e., aimed to increase or maintain one or more components of physical fitness). It is considered as a subcategory of physical activity. older adults, at least 150 min/week of moderate-to-vigorous physical activity or 75 min/ week of vigorous physical activity. For children and adolescents, 60 min or more of moderate-to-older adults, limit the amount of time spent sedentary and replace sedentary time with more physical activity of any intensity. For children and adolescents, limit the amount of time spent sedentary particularly recreational screen time.

  as measured by area under the curve; ?, no data available; NA, not applicable; NS, non-significant. Abbreviations: FMD, flow-mediated dilation; HDL, highdensity lipoprotein; LDL, low-density lipoprotein; SMD, standard mean difference. Data presented as mean difference (95% confidence interval) between reducing/interrupting sedentary behavior and prolonged sedentary behavior/control group.

FIGURE 1 .

 1 FIGURE 1. Sedentary behavior and physical inactivity operationalized as distinct

FIGURE 2 .FIGURE 3 .

 23 FIGURE 2. Hypothetical representation of imposed changes in physical activity level by

FIGURE 4 .

 4 FIGURE 4. Mechanisms underlying sedentary behavior-and interruptions to sitting-

FIGURE 5 .

 5 FIGURE 5. Vascular responses following exposures to prolonged sedentary behavior

FIGURE 6 .

 6 FIGURE 6. Key anabolic and catabolic pathways in muscle atrophy conditions. Several

FIGURE 7 .

 7 FIGURE 7. Cerebrovascular responses following exposures to prolonged sedentary

TABLE OF CONTENTS

 OF

Table 1 .

 1 T, Kornips E, Hulsbosch M, Willems P, Hesselink MKC, Schrauwen P, Savelberg H. Benefits of Substituting Sitting with Standing and Walking in Free-Living Conditions for Cardiometabolic Risk Markers, Cognition and Mood in Overweight Adults. Front Physiol 8: 353, 2017. doi: 10.3389/fphys.2017.00353. 123. Duvivier BM, Schaper NC, Bremers MA, van Crombrugge G, Menheere PP, Kars M, Savelberg HH. Minimal Intensity Physical Activity (Standing and Walking) of Longer Duration Improves Insulin Action and Plasma Lipids More Than Shorter Periods of Moderate to Vigorous Exercise (Cycling) in Sedentary Subjects When Energy Key terms and definitions in sedentary behavior and physical inactivity research.

	Expenditure	Is	Comparable.	PLoS	One	8:	e55542,	2013.	doi:
	10.1371/journal.pone.0055542.
	124. De Jong NP, Rynders CA, Goldstrohm DA, Pan Z, Lange AH, Mendez C,
	Melanson EL, Bessesen DH, Bergouignan A. Effect of Frequent Interruptions of
	Sedentary Time on Nutrient Metabolism in Sedentary Overweight Male and Female
	Adults.	J	Appl	Physiol	(1985)	126:		984-992,	2019.	doi:
	10.1152/japplphysiol.00632.2018.
	125. Blankenship JM, Winkler EAH, Healy GN, Dempsey PC, Bellettiere J, Owen N,
	Dunstan DW. Descriptive Epidemiology of Interruptions to Free-Living Sitting Time in
	Middle-Age and Older Adults. Med Sci Sports Exerc 53: 2503-2511, 2021. doi:
	10.1249/MSS.0000000000002750.

TABLE 2 .

 2 Sedentary behavior frequency, intensity, time, and type principle.Frequency Refers to the number of SB bouts over a given time frame. The most common SB bout lengths reported in the literature are ≥30 minutes, ≥60 minutes, and ≥120 minutes. Example: Interruptions to sitting lasting 2-3 minutes every 20-30 minutes seems to yield greater benefits for glycemic control compared to prolonged, uninterrupted sitting.Study design, data analysis, and reporting: Include experimental groups with different frequency of interruptions to SB versus a more sedentary experimental group (control group). If possible, groups should be matched for duration and/or EE. Include detailed description about how participants were instructed about the frequency of interruptions and how adherence to prescribed frequency was assessed. Report changes in the number of daily interruptions to SB and number and duration of prolonged SB bouts.IntensityRefers to any waking behavior with an energy expenditure ≤1.5 MET, while sitting, reclining, or lying down. When focusing at reducing/ interrupting sedentary behavior, intensity refers to the physical activity used to replace sedentary behavior. Example: EE during sitting postures is lower than standing and all intensities of physical activity. Similar responses are observed for oxygen consumption and heart rate. Study design, data analysis, and reporting: Include experimental groups with different intensity of interruptions to SB versus a more sedentary experimental group (control group). If possible, groups should be matched for EE. Include detailed description about how participants were instructed about the intensity of interruptions and how adherence to prescribed intensity was assessed. Report changes in objectively measured daily time spent in each physical activity intensity. total duration of time spent in SB or time spent in bouts of uninterrupted, prolonged sitting (i.e., consecutive minutes accumulated in SBusually reported as bouts of ≥30 minutes, ≥1 hour and ≥2 hours of sitting). Example: Increasing sedentary time results in maladaptations in physiological systems. In contrast, reducing/interrupting sedentary time results in small benefits. Study design, data analysis, and reporting: Include detailed description about how participants were instructed to reduce/interrupt SB (as per other FITT elements) and how adherence to prescribed intervention was assessed. If possible, provide participants with specific and measurable goals, so adherence can be objectively assessed. Report changes in duration of objectively measured total daily SB and prolonged SB bouts. Type Refers to the main intention of the SB and the context in which it occurs. Example: SB associated with energy surplus is more detrimental than exposures to sedentary behavior in energy balance. Study design, data analysis, and reporting: Measure participation in each type of SB using a validated questionnaire or diary. Similarly, record types of physical activity that can be used to reduce/interrupt SB. If appropriate, design the intervention to tackle specific type/context of SB (e.g., workrelated, recreational screen time, etc.) and standardize the FITT of physical activities being used to reduce/interrupt SB. Report changes in duration of objectively measured total daily SB and prolonged SB bouts, and self-reported duration in each specific type/context of SB.

	Term	Definition

TABLE 3 .

 3 Summary of acute (hours to <4 days) and longer-term (>2 weeks) effects (experimental/intervention) of increasing sedentary behavior in adults and older adults. : *, protocols involved resistance exercise alone or resistance and aerobic exercise training. In general, combined exercise training was more effective at attenuating or counteracting adverse effects related to bed rest than resistance exercise training alone.

					Acute effects		Longer-term effects
	Outcomes
					Mean change	Refs.	a	Mean change	Refs.	a
	Body mass and composition
	Body weight (kg)				NA	-	-2%	(108)
	Total and percentage body fat (%)	NA	-	3 to 14%	(106, 107,
							128)
	Intra-abdominal fat mass (%)		NA	-	7%	(108, 109)
	Glucose metabolism
	Fasting glucose (mg/dL)			NA	-	NS	(108, 147)
	2-hour post-load glucose (mg/dL)	?	-	NS	(147)
	Postprandial glucose (%)	b		17.5 (-26.2 to -8.7)	(75)*	6 to 9%	(117)
	Glycemic variability				NA	-	33 to 97%	(117)
	Whole-body insulin sensitivity (%)	-39 to -18%	(162)	-17%	(147)
	Fasting insulin (μU/mL)				NA	-	NS	(108, 147)
	2-hour post-load insulin (μU/mL)	?	-	38.8 μU/mL	(147)
	Postprandial insulin (%)	b			25.1 (-31.8 to -18.3)	(75)*	32%	(147)
	Lipid metabolism
	Fasting triglycerides (mg/dL)		NA	-	0.3 mmol/L	(106, 112)
	Postprandial triglycerides (%)	b	27%	(72)	21%	(109)
	Fasting total cholesterol (mmol/L)	NA	-	0.5 mmol/L	(112)
	Fasting LDL (mmol/L)				NA	-	0.3 mmol/L	(112)
	Fasting HDL (mmol/L)				NA	-	-0.1 mmol/L	(106)

Legend208

TABLE 5 .

 5 Summary of acute (<24 hours) and longer-term (>2 weeks) beneficial effects (experimental/intervention) of reducing/interrupting sedentary behavior in adults and older adults, as evidenced by meta-analyses.

	Acute effects		Longer-term effects
	Outcomes
	Mean difference	Refs.	Mean difference	Refs.

Acknowledgements

We are extremely grateful for the resourcefulness and hard work of our broader network of collaborators and the research fellows, PhD students, and support staff who have worked with us in our programs of sedentary behavior research. Figures were created with Mind the Graph (mindthegraph.com). Grants A.J.P. and B.G. were supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Grants 2015/26937-4 and 2018/19418-9, and 2017/13552-2). A.J.P. received funding from the Ludeman Family Center for Women's Health Research at the University of Colorado Anschutz Medical Campus. A.B. were supported by the National Institutes of Health (NIH NIDDK, Grant R01DK123334). D.W.D., P.C.D. and N.O. were supported by the National Health and Medical Research Council of Australia (NHMRC) Centre of Research Excellence (Grants #1057608 and #1142685), by the Victorian state Government Operational Infrastructure Support scheme, and by the NHMRC Fellowships scheme.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the authors.

Correspondence

Prof David Dunstan (e-mail: David.Dunstan@baker.edu.au).

Authors contributions

Musculoskeletal system

Lean mass (%) NA --9 to -0.5% [START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF](107)(108)(109)337) Muscle strength (%) NA --31 to -8% [START_REF] Suetta | Effects of Aging on Human Skeletal Muscle after Immobilization and Retraining[END_REF]130,337,356) Bone mineral density (%) NA -0.3 to -1% (365)

Central nervous system

Cerebral artery blood flow

Fat mass and repartition

Intra-abdominal fat mass increases counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]247) Liver fat accumulation increases attenuated ( 34)

Glucose metabolism

Whole-body insulin sensitivity decreases attenuated to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]170,244) Fasting insulin increases no effect to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]170,244) Fasting carbohydrate oxidation increases no effect to attenuated [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Postprandial carbohydrate oxidation increases no effect to counteracted (34, 244)

Lipid metabolism

Fasting triglycerides increases no effect [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Fasting HDL decreases no effect [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Fasting lipid oxidation decreases no effect to attenuated [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]170,244) Postprandial lipid oxidation decreases no effect to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]244) Cardiorespiratory fitness VO 2max decreases attenuated to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF]406)

Musculoskeletal system

Muscle mass decreases attenuated to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Muscle fiber type toward glycolytic increases attenuated [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Mitochondrial oxidative capacity decreases attenuated [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Muscle strength decreases counteracted [START_REF] Gallagher | Effects of 84-Days of Bedrest and Resistance Training on Single Muscle Fibre Myosin Heavy Chain Distribution in Human Vastus Lateralis and Soleus Muscles[END_REF][START_REF] Trappe | Human Single Muscle Fibre Function with 84 Day Bed-Rest and Resistance Exercise[END_REF]407) Muscle fat storage increases no effect (170)

Bone mineral density decreases attenuated or counteracted (408,409) Bone fat storage increases no effect to counteracted [START_REF] Roux | Physiology of Physical Inactivity, Sedentary Behaviours and Non-Exercise Activity: Insights from the Space Bedrest Model[END_REF] Inflammatory responses