# Impact of Long-Term Fasting on Skeletal Muscle: Structure, Energy Metabolism and Function Using 31 P/ 1 H MRS and MRI

Antoine Naëgel, Magalie Viallon, Hélène Ratiney, Thu Nguyen, Benjamin Leporq, Djahid Kennouche, Thomas Grenier, Franziska Grundler, Robin Mesnage, Jean-michel Guy, et al.

► To cite this version:

Antoine Naëgel, Magalie Viallon, Hélène Ratiney, Thu Nguyen, Benjamin Leporq, et al. Impact of Long-Term Fasting on Skeletal Muscle: Structure, Energy Metabolism and Function Using 31 P/ 1 H MRS and MRI. Journal of Cachexia, Sarcopenia and Muscle, 2025, 16 (2), pp.e13773. 10.1002/jcsm.13773. hal-05132099

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<table><tr><td>Manuscript Number:</td><td></td></tr><tr><td>Full Title:</td><td>Impact of long-term fasting on skeletal muscle: structure, energy metabolism, and function using 31P/1H MRS and MRI</td></tr><tr><td>Article Type:</td><td>Original Article</td></tr><tr><td>Corresponding Author:</td><td>Pierre Croisille, PUPH, MD, PhD<br>CHU Saint-Étienne<br>Saint-Étienne, FRANCE</td></tr><tr><td>Corresponding Author Secondary Information:</td><td></td></tr><tr><td>Corresponding Author's Institution:</td><td>CHU Saint-Étienne</td></tr><tr><td>Corresponding Author's Secondary Institution:</td><td></td></tr><tr><td>Corresponding Author E-Mail:</td><td>pierre.croisille@creatis.insa-lyon.fr</td></tr><tr><td>First Author:</td><td>Antoine Naegel, PhD</td></tr><tr><td>First Author Secondary Information:</td><td></td></tr><tr><td>Order of Authors:</td><td>Antoine Naegel, PhD<br>Magalie Viallon, PhD<br>Hélène Ratiney, PhD<br>Thu Nguyen, PhD<br>Benjamin Leporq, PhD<br>Djahid Kennouche, PhD<br>Thomas Grenier, PhD<br>Franziska Grundler, PhD<br>Robin Mesnage, PhD<br>Jean-Michel Guy, MD<br>Françoise Wilhelmi de Toledo, MD<br>Pierre Croisille, MD, PhD</td></tr><tr><td>Order of Authors Secondary Information:</td><td></td></tr><tr><td>Abstract:</td><td>Background Fasting is a promising tool for public health, but concerns about muscle loss prevent its acceptability in mainstream medical practice, especially in older persons. This study explored the impact of long-term fasting (12 days, 250kcal/day) on muscle structure, metabolism, and performance.<br>Methods In a prospective study of 32 subjects (16 men and 16 women; 16≤ 50 and 16>50 years-old), muscle volume, composition, relaxometry data, and lipid metabolism were assessed before, at the end, and one month after fasting. Assessments involved high-resolution 3D Dixon anatomical MR imaging, multi-echo CSE acquisitions, and single-voxel MR spectroscopy. Dynamic 31P-MRS acquisitions, quantitative MRI, maximal voluntary contraction (MVC) measures, and cardiorespiratory fitness (VO2max) were repeatedly examined throughout the protocol.<br>Results Average decrease of weight and skeletal muscle volumes was 5.9kg (7.4%) and 542ml</td></tr></table>
<table><tr><td></td><td>(5.4%), respectively. This closely aligns with glycogen (1-2%) and bound water losses (3-4%) that are expected to reach 404-505 ml. MVC (anaerobic lactic metabolism) remained preserved in both upper and lower legs, regardless of sex or age. Unchanged T2 showed fasting did not induce structural or inflammatory changes. MRI/MRS revealed fat redistribution among tissues, with subcutaneous fat decrease and total fat fraction increase in muscle. Intramyocellular lipid pool increased while the extracellular lipid pool decreased revealing rapid lipid trafficking and rapid adaptation. The T2* increase during fasting likely stems from changes in intracellular lipid droplets configuration, with an increased proportion of lipid droplets of smaller size, optimizing accessibility of lipid fuels and mitochondrial FA. Cardiopulmonary exercise testing (VO2 max) revealed no alteration in maximal oxygen uptake during fasting, but an improved fat oxidation capability with a 10% increase in the first ventilatory threshold and a 10% decrease in the respiratory exchange ratio. Mitochondrial oxidative capacity and the rate of PCr resynthesis in muscle were maintained. Females improved their mitochondrial function at D+12, surpassing males, expressing better fat oxidation capabilities.<br/><br/>Conclusion:<br/>Long-term fasting did not alter muscle metabolism or performance, nor induced structural or inflammatory changes. Altogether, the leg muscle decrease volume is small when considering glycogen + water depletion during fasting. Fat relocates in the intracellular compartment of myocytes. Anaerobic and aerobic metabolic pathways remain unchanged after 12-day fasting in both sexes and older subjects. This suggested that human muscles, like those of animals, were evolutionary adapted by seasonal food shortages to sustain long-term fasting.</td></tr><tr><td>Suggested Reviewers:</td><td>Benjamin Marty, PhD<br/>Institute of Myology<br/>b.marty@institut-myologie.org<br/>expertise in 31P NMR spectroscopy for non-invasive analysis of muscle metabolism, to understand intramyocellular energetics, oxidative capacity, and lipid stores during disease and lifestyle interventions.<br/><br/>Audrey Bergouignan, PhD<br/>Associate Professor, University of Colorado Anschutz Medical Campus School of Medicine<br/>audrey.bergouignan@gmail.com<br/>integrative physiologist with expertise in metabolic flexibility and adaptation, integrating lifestyle environmental signals to quantify their impact on energy balance and metabolic control systems<br/><br/>Ellen E Blaak, PhD<br/>Professor, Maastricht University NUTRIM School for Nutrition Toxicology and Metabolism<br/>e.blaak@maastrichtuniversity.nl<br/>expertise in nutrition and metabolism, including the mechanisms of substrate oxidation, endocrine and enzymatic regulation around fat oxidation, carbohydrate oxidation and protein turnover during exercise and diet changes</td></tr><tr><td>Author Comments:</td><td>While we are perfectly aware of the 40 references limit (recommandations to authors), we intend to provide final bibliography referencing, once the reviewer process is done, not only because it eases their reading, but also because it will facilitate to add or remove any references while following their recommandations or requests. We hope you understand.</td></tr></table>
Impact of long-term fasting on skeletal muscle: structure, energy
metabolism, and function using 31P/1H MRS and MRI

Antoine Naëgel¹, Magalie Viallon ¹,², Hélène Ratiney¹, Thu Nguyen¹, Benjamin Leporq¹, Djahid Kennouche³, Thomas Grenier¹, Franziska Grundler⁴, Robin Mesnage⁴,⁵, jean-Michel Guy⁶, Robin Schultz⁷, Françoise Wilhelmi de Toledo⁴ and Pierre Croisille¹,²

¹Univ Lyon, UJM-Saint-Etienne, INSA, CNRS UMR 5520, INSERM U1206, CREATIS, F-42023, Saint-Etienne, France

²Department of Radiology, University Hospital Saint-Etienne, Saint-Etienne, France

³Université Jean Monnet Saint-Etienne, Lyon 1, Université Savoie Mont-Blanc, Laboratoire Interuniversitaire de Biologie de la Motricité, F-42023, Saint-Etienne, France

⁴Buchinger Wilhelmi Clinic, Wilhelmi-Beck-Straße 27, 88662 Überlingen, Germany

⁵ Department of Nutritional Sciences, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, SE1 9NH London, UK

⁶ Le Clos Champirol Reeducation, 81 avenue Albert Raimond, 42270 Saint-Priest-en-Jarez, France

⁷ Mettnau, Werner-Messmer-Klinik, Strandbadstr. 80, 78315 Radolfzell am Bodensee, Germany

**Corresponding Author:**

pierre.croisille@creatis.insa-lyon.fr

Phone: sec: +33477829028 /direct: +33477127584
**Abstract**

**Background**

Fasting is a promising tool for public health, but concerns about muscle loss prevent its acceptability in mainstream medical practice, especially in older persons. This study explored the impact of long-term fasting (12 days, 250kcal/day) on muscle structure, metabolism, and performance.

**Methods**

In a prospective study of 32 subjects (16 men and 16 women; 16≤ 50 and 16>50 years-old), muscle volume, composition, relaxometry data, and lipid metabolism were assessed before, at the end, and one month after fasting. Assessments involved high-resolution 3D Dixon anatomical MR imaging, multi-echo CSE acquisitions, and single-voxel MR spectroscopy. Dynamic 31P-MRS acquisitions, quantitative MRI, maximal voluntary contraction (MVC) measures, and cardiorespiratory fitness (VO2max) were repeatedly examined throughout the protocol.

**Results**

Average decrease of weight and skeletal muscle volumes was 5.9kg (7.4%) and 542ml (5.4%), respectively. This closely aligns with glycogen (1-2%) and bound water losses (3-4%) that are expected to reach 404-505 ml. MVC (anaerobic lactic metabolism) remained preserved in both upper and lower legs, regardless of sex or age. Unchanged T2 showed fasting did not induce structural or inflammatory changes. MRI/MRS revealed fat redistribution among tissues, with subcutaneous fat decrease and total fat fraction increase in muscle. Intramyocellular lipid pool increased while the extracellular lipid pool decreased revealing rapid lipid trafficking and rapid adaptation.

The T2* increase during fasting likely stems from changes in intracellular lipid droplets configuration, with an increased proportion of lipid droplets of smaller size, optimizing accessibility of lipid fuels and mitochondrial FA. Cardiopulmonary exercise testing (VO2 max) revealed no alteration in maximal oxygen uptake during fasting, but an improved fat oxidation capability with a 10% increase in the first ventilatory threshold and a 10% decrease in the respiratory exchange ratio. Mitochondrial oxidative capacity and the rate of PCr resynthesis in muscle were maintained. Females improved their mitochondrial function at D+12, surpassing males, expressing better fat oxidation capabilities.
**Conclusion:**

Long-term fasting did not alter muscle metabolism or performance, nor induced structural or inflammatory changes. Altogether, the leg muscle decrease volume is small when considering glycogen + water depletion during fasting. Fat relocates in the intracellular compartment of myocytes. Anaerobic and aerobic metabolic pathways remain unchanged after 12-day fasting in both sexes and older subjects. This suggested that human muscles, like those of animals, were evolutionary adapted by seasonal food shortages to sustain long-term fasting.

**Keywords:** Long-term fasting; muscle metabolism; substrate utilization; muscle strength; magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS)
Introduction

Human life expectancy has steadily increased in the past centuries thanks to improved medical care. However, the modern unhealthy Western lifestyle has hindered this progress by promoting an epidemic of chronic metabolic and inflammatory diseases¹. This has led to research into therapeutic strategies that promote healthy aging, with fasting (intermittent and long-term) and calorie restriction diets emerging as the most promising approaches to extend healthy lifespan ²,³. Fasting has always been practiced in free-living animals to cope with seasonal changes in food availability, and in humans for religious and spiritual reasons. However, the development of fasting as a therapeutic approach has led to a debate on a possible muscle loss ⁴,⁵. This is because the body can mobilize proteins to supply amino acids to the liver and kidney for neoglucogenesis, ensuring that the brain and other organs receive the glucose they need to function normally.

Concerns about loss of skeletal muscle during fasting mainly originate from studies conducted in the 1960s on the energy metabolism of obese individuals undergoing long periods of water-only fasting for weight loss⁶,⁷. Whether fasting affects muscle amino acid metabolism remains controversial as some studies report that net muscle amino acid release increases with fasting⁸, while others report this is unaltered. In addition, while studies have shown that the body metabolizes amino acids during fasting, it is unclear whether these amino acids come from the muscles and other organs that are profoundly remodeled by autophagy⁹. Furthermore, studies showing that protein use can be different between lean subjects and obese subjects, and according to the duration of the fasting period, prompt the need for new studies in healthy subjects from both sexes and different age groups ⁵.

The density of proteins of the muscular tissue has long lead to the assumption that the protein utilization for neoglucogenesis which is maximal in the first days of fasting was provided by the mobilization of proteins from skeletal muscles. Natural cycling in muscle mass between periods of eating and fasting is described in wild animals such as king penguins¹⁰. In this case, protein usage during long periods of fasting remains limited due to the activation of protein-sparing mechanisms when ketone bodies are generated from fatty acids¹¹. Muscle mass loss is recovered when the animal is re-fed, as reflected by

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decreased myostatin levels in fasting animals. The same is observed in humans after the food reintroduction ⁴. The reversibility of these modifications suggests that any changes in muscle metabolism in fasting humans could only be transitory adaptations to the fasting metabolism. We hypothesize that amino acids utilized to produce glucose during fasting can come from other tissues through autophagy. This is clear in laboratory rodents for which it is showed that liver-specific autophagy provided amino acids for neoglucogenesis to maintain blood glucose levels¹². As a matter of fact, no study has explored whether any effect observed on muscle composition in fasting humans is a permanent damage or a reversible effect.

The safety of natural variations in muscle mass in animals during seasonal variations in food availability further suggests that there is a need to look at changes beyond composition to understand if the function of the muscles is affected. No studies have examined whether fasting affects muscle metabolism, performance, or integrity beyond muscle composition. Only a recent prospective trial in healthy men confirmed a protein loss occurring in early fast, decreasing as ketogenesis increases, but concluded that fasting combined with physical activity does not seem to negatively impact muscle function ⁴. Thus, new studies using advanced non-invasive technologies are needed to investigate the impact of fasting on the human body with a particular focus on skeletal muscle and improve our understanding of both possible functional and compositional changes. Dynamic 31P-Magnetic Resonance Spectroscopy (31P-MRS) and proton MR Imaging (1H-MRI) can offer unrivaled non-invasive longitudinal quantification capabilities of organ changes from macroscopic anatomy to microscopic metabolic changes and function in muscle, as performed in the CALERIE 2 study of the effects of caloric restriction on mitochondrial function ¹³,¹⁴.

In this prospective longitudinal study, we use quantitative MRI and MRS to investigate macroscopic to metabolic muscle coupled changes in a representative population of healthy men and women before, at the end of a 12-day fast and physical activity program, and one month after food reintroduction.

**Materials and Methods**

*MR protocol and studied population*
High-resolution anatomical 3D isotropic Dixon sequence, quantitative T2 mapping, 1H, and dynamic 31P-MRS acquisitions were performed on the quadriceps and calf weight-bearing on a 3T clinical MRI at three time points: before (D-1), at the end (D+12), and one month (M+1) after the end of fasting in 16 men and 16 women (44 ± 14 years; 26.2 ± 0.9 kg/m2) who fasted with a supplement of 250 kcal/day and maintained up to 3 h daily low-intensity physical activity in the GENESIS (lonG tErm FastiNg multi-systEm adaptationS In humanS) study: a prospective, monocentric, single-arm interventional study¹⁵. The fasting protocol was as described in peer-reviewed guidelines. Table S1 in supplementary material summarizes the main parameters of the MR sequences that constitute the MR protocol ran at each time point of the interventional study. Briefly, a 3D gradient multi-echo chemical-shift-encoded imaging (CSE-MRI) sequence was used to extract fat fraction map and relaxometry data¹⁶. We derived lipid metabolism from single-voxel STEAM 1H-MRS acquisitions on the thigh muscle. We explored energy metabolism by dynamics FID 31P-MRS acquisitions on volunteers' legs with saturation bands to minimize the signal from unstressed muscle and bone. The platform was equipped with an MRI-compatible ergometer (ErgoSpect GmbH, Innsbruck, Austria) with its calf module. Subjects were lying supine with the dual 1H/31P surface coil under their calf muscle (Figures 1 and 2).

## Activity monitoring, muscular strength measurement and Cardiopulmonary exercise testing

Activity monitoring was quantified continuously starting two weeks before fasting until two weeks after re-feeding. The quadricipital and calf maximal voluntary contraction (MVC) were respectively measured on the same day of each MR session (D-1, D+12, and M+1). A maximal cardiopulmonary exercise cycle ergometer test was also conducted at each MR session (See supplementary materials for details).

## Muscle volume, metabolite concentrations, and time constants quantification

Lower and upper leg muscle volume were obtained using automatic segmentation process on T1 Dixon Water-only (T1W) and Fat-only (T1F) 3D MR images using a multi-atlas segmentation with joint
label fusion¹⁷,¹⁸ in all 32 subjects at the three observation time points. T2 in the calf muscle was further extracted from the T2 maps as described by Nguyen et al. ¹⁷.

1H-MRS metabolites were quantified after fitting the acquired spectra using the standard LCModel¹⁹, to extract IMCL<sub>CH2</sub> resonances (IMCL at 1.3ppm), EMCL<sub>CH2</sub> (EMCL at 1.5ppm), and Taurine (Tau) at 3.4ppm. We also used Choline (Cho) to refer to any Trimethylammonium (TMA) compounds as proposed by others²⁰. We calculated relative concentrations for all metabolites using the Creatine resonance (Cr) at 3.0 ppm as a reference²¹.

We calculated 31P-MRS metabolites concentrations and associated time constants in agreement with the latest recommendations²²⁻²⁴. Quantification was performed using the QUEST (QUantitation based on QUantum ESTimation) method²⁵. The millimolar concentration of phosphorus metabolites was evaluated based on the standard assumption that [ATP] is 8.2 mmol/L of cell water²⁶. The concentration of the metabolites PCr, Pi, ATP, ADP, and pH were extracted at rest, post-exercise, and after recovery.
We also calculated the PCr depletion (percentage) during plantar exercise, the time constants of PCr (τPCrEx and τPCrRec) and Pi (τPiEx and τPiRec) during exercise, during recovery, as well as the initial recovery rate of PCr (ViPCr) and the maximum aerobic capacity (Vmax) according to ²²,²⁷. During recovery, τPCrRec reflects the rate of PCr resynthesis and mitochondrial oxidative capacity to regenerate ATP, with lower values meaning faster recovery kinetics and higher oxidative capacity.
ViPCr measures PCr recovery kinetics; faster PCr recovery kinetics means improved maximal mitochondrial ATP production. The maximum aerobic capacity (Vmax) represents the theoretical maximum velocity at which muscle can synthesize ATP via oxidative phosphorylation and is dependent on the number and function of mitochondria in muscles.

## Statistics

All variables were screened for normality using the Shapiro-Wilk test and reported as the mean (SD) or mean [95%CI] when appropriate. To examine the effects of age and sex (between-subjects factors) and time (within-subjects factor) and their interactions, we used a 3-way repeated-measures mixed-effects model approach with a compound symmetry covariance matrix and REstricted Maximum
Likelihood (REML) fitting. A Geisser-Greenhouse correction was applied to account for the sphericity assumption violation. When applicable, pairwise post-hoc comparisons were obtained with a Tukey test to correct for multiple comparisons. For all analyses, significance was accepted at p< 0.05. The statistical analyses were conducted using Prism 10.1 (La Jolla, CA, USA) and Stata 18 (College Station, TX).

## Results

### Demographic data and total body weight changes

Baseline anthropometric data on the subject's characteristics are given in Table S2 in supplementary material. As expected, fasting led to a significant weight loss (p=0.002) without a significant effect of sex or age (p=0.12 and p=0.93, respectively) and no two-way interactions. The average total body weight loss from baseline was -5.9 [-6.4;-5.5]kg at D+12 and -4.1[-4.8;-3.3]kg at M+1 (Table 1).

### Muscular volumes and strength measurements

Results are detailed in Table 1. We observed a significant effect of time (fasting) on upper leg muscular volumes (p<0.0001), as well as an effect of sex (p<0.0001) but no influence of age (p=0.068) (see also Figure 3). On lower leg muscular volumes, we observed a similar pattern but with a time * sex interaction (p<0.007) and no influence of age on leg muscle volumes (p=0.23). Overall, the upper leg muscular volume difference between males and females was +1497ml, while the lower leg volume difference was +383ml (all p<0.0001). At D+12, upper leg muscle volume loss was -270[-355,-184]ml in male (p<0.0001) and -165[-211,-119]ml (p<0.0001) in female. Between D+12 and M+1, the volume gain was relatively more significant in female +75.6[9,142]ml (p=0.02) than in male +102[-13,221]ml (p=0.08). Lower leg volume loss at D+12 was -77[-103,-50]ml (p<0.00001) in male and -39[-61,-17]ml (p=0.0008) in female. Between D+12 and M+1, male gained +33[2,64]ml (p=0.035) while female remain unchanged with -2.3[-13.7,9.13]ml, p=0.86. The average total muscle volume loss at D+12 (thighs+lower legs, assuming a R/L symmetry) was overall 542ml (-5.4% versus baseline), 690ml (-5.7%) in males and 406ml (4.9%) in females. After re-feeding at M+1, muscle volume difference remained overall at 336ml (3.5%), 376ml (3.3%) in males, and 262ml (3.3%) in females (Table 2).
Muscle strength evaluated by maximum voluntary contraction (MVC) measures was stable over time and fasting for upper and lower legs (p=0.138 and p=0.089, respectively) (Table 1). We observed
significant differences between males and females (upper legs: p=0.0003 and lower legs: p=0.016, respectively). In contrast, a difference between the young and aged groups existed only for upper leg measures (p=0.01), without any two-way interactions. All MVC measures increased at D+12 compared to baseline reference values (all p=NS), with a +4.6% in aged and +2.4% in young subjects, +4.2% in male and + 1.6% in female sub-groups when considering upper leg measures (Table 1).

## Activity monitoring and cardiopulmonary exercise testing

The amount of vigorous activity was unchanged among the different time points (p=0.36) and was therefore maintained during fasting by the exercise program, without any sex (p=0.47) or age (p=0.41) differences (see Table S3). On the other hand, we observed changes in the breakdown of lower activity levels, with moderate and light activity levels decreasing during fasting (p<0.001). In contrast, the sedentary activity level increased during fasting (p<0.002).

All details of peak exercise capacity testing and parameters are in Table S4, and Figure 4. Maximal oxygen uptake (VO2max) was remarkably stable over time (p=0.669), without any decrease at D+12 compared to baseline. There were no statistically significant two-way interactions or age and sex effects. Ventilatory threshold (VT1 and VT2) changes over time also relied on age, as illustrated by the significant time*age interactions (p=0.0257 and p=0.042, respectively). Compared to baseline, VT1 increased more in younger subjects at D+12 (+15%; p=0.022) than older ones (+8%; p=0.529) at M+1 and compared to baseline (-9.2%, p=0.669), while older subjects further increased VT1 (+15.8%, p=0.122).

The respiratory exchange ratio (RER) varied significantly over time (p<0.001), with a significant reduction at D+12 and with a time *sex interaction (p=0.017). Indeed, while males and females had similar RER ratios at baseline (p=0.916) and M+1(p=0.466), the RER ratio decreased significantly more at D+12 in females (-12.7% from baseline) than in males (-5.7%) (p=0.0095) (see Figure 5).

## MR Relaxometry and upper-leg tissues fat composition

Table S5 provides all the details regarding the changes in skeletal muscle, subcutaneous adipose tissue (SAT), and bone marrow adipose tissue (BMAT) composition at the upper leg level.
In skeletal muscle, relaxometry measures showed no changes in T2 but a significant T2* increase over time (p<0.0001) ceiling at D+12, together with significant differences related to age (p=0.0145). In parallel, fat fraction (PDFF%) in muscle increased at the end of fasting (p=0.002) and more significantly in older individuals (+22% at D+12) (p=0.019). 1H-MRS provides further understanding of muscle content changes: IMCL exhibited time variations (p=0.009), with a +113% average increase at the end of fasting (p=0.019), while returning to baseline at M+1. At the same time, EMCL decreased at D+12 (p=0.012, -28%) while remaining significantly lower at M+1 than at baseline (p<0.0001) (see Figure 6A). There were no significant changes in Taurine and Choline metabolites in muscle tissue.

In SAT, T2* and PDFF both decreased during time (both p<0.0001), with a sex\*time interaction and PDFF lowering more in males and at M+1 (p=0.005). The fat composition also changed until M+1 with more SFA, and less MUFA and PUFA (p<0.0001) and sex differences (p<0.0001). In BMAT, there were no T2* and PDFF changes over time, with more transient changes in all fat composition (all p <0.05).

## 31P-MRS derived metabolites and time constants in muscle

Table S6 provides the main variables quantified by dynamic 31P-MRS (rest, during exercise, and recovery) assessing the muscular energy balance at the different time points of the study (see also Table S7 for additional results). There were no significant differences in high-energy phosphate intermediates, [PCr] and [Pi] remaining stable over time. The pH was significantly lowered at the end of fasting, existing at rest (p<0.0001), but acidosis did not worsen during exercise (p=0.0016) or recovery. Of note, there were sex differences with lower pH in females at rest (p=0.015) and recovery (p=0.010) pre-existing at baseline. The PCr depletion/consumption during aerobic exercise was comparable between the fed and unfed phases and without differences across sex and age groups. Overall, mitochondrial oxidative capacity remained stable over time, but with sex differences related to (p=0.02) with a faster recovery in females, especially at end-fasting (p=0.004) (Figure 6B). Note that $\tau$PiRec showed changes over time and increased at end-fasting (0.007).
Discussion

The acceptability of therapeutic fasting by conventional medicine has been notably hindered by concerns
about its impact on skeletal muscle, questioning muscle loss, and, ultimately, sarcopenia, implying that
older subjects would be more exposed to this risk. Nevertheless, this has never been scientifically
investigated in detail. Our study showed that, although muscle volume decreased to some extent after
12 days of fasting, skeletal muscle metabolism and function were maintained without any deleterious
effects even in older subjects. Importantly, these outcomes remained consistent even one month after
fasting while muscle volume restoration was underway.

Skeletal muscle volume and MVC force measures

The leg's total skeletal muscle volume loss at D+12 (end-fasting) of 542ml (-5.4%) in the whole
population should be put in perspective with the expected changes that are directly related to glycogen
storage depletion. Such changes occur within a few days during fasting but also during dieting.

In sedentary untrained subjects adhering to a regular mixed diet, the average skeletal muscle glycogen
storage is 1-2% of wet muscle weight. However, factors such as diet and activity can elevate this
percentage to 4-5% in highly active individuals 28,29. Additionally, every gram of glycogen is bound to
an estimated 3-4 grams of water due to direct molecular bounding and osmosis30,31. This leads to
significant weight changes that depend only on glycogen storage changes. Considering a glycogen
content loss of 1%, the calculated volume loss related to the glycogen depletion and the bounded water
(3-4%) would be 404-505 ml. Therefore, the estimated volume loss corresponding to the combination
of glycogen depletion and bounded water volume in the leg's skeletal muscle is close to the volume loss
quantified in our study. This indirect assessment suggests that the actual protein loss in skeletal muscle
related to neoglucogenesis is likely minimal.

An essential finding of the study is the total preservation of the leg's muscle function quantified by the
MVC measures at the end of the fasting period (D+12). This observation indicates that the high-intensity
anaerobic alactic metabolism pathway mainly related to fast-twitch fiber recruitment is operational and
intact, and also suggested the absence of impairment in the neural CNS output or central fatigue at the
end of fasting³².
Cardiorespiratory exercise testing findings

The maximal cardiopulmonary exercise ramped testing was used to quantify the overall ability to deliver oxygen to exercising skeletal muscles and of the exercising muscles to extract oxygen. This latter point is addressed explicitly by 31P-MRS measures discussed below.

Our study found that the maximum oxygen uptake capacity (i.e., VO2 max) stayed remarkably stable over time and was maintained at the end of the fasting period, while the maximum power output at peak exercise was also maintained.

We also documented an improvement of the first ventilatory threshold (VT1) which is of practical interest from a functional point of view. From a physiological exercise standpoint, VT1 is associated with the initial rise and commencement of blood lactate accumulation. However, for non-athlete sedentary or active subjects, this translates to a better ability to achieve daily tasks, including walking, that are for their majority performed below submaximal exercise level. Therefore, increasing this VT1 would allow one to perform everyday activities at a lower relative level of the maximal level and thus limit the impact on fatigue <sup>33</sup>. The last important finding is the observation of a decrease in the RER at the end of fasting. A decrease of the RER during an exercise test reflects a greater reliance on fat oxidation and utilization of plasma-free fatty acids for energy production while signing (if needed) the muscular carbohydrate stores depletion<sup>34,35</sup>. While we found no difference in the response between age groups, we found that females had lower RER in female but only after fasting. While most studies comparing substrate oxidation among genders describe lower RER in females, Tarnopolsky discussed that a lack of difference may be related to confounding factors (diet, body composition, time since last exercise boot, level of fitness, menstrual phase, or amenorrhea)<sup>36</sup>. Therefore, if the lack of difference in RER at baseline between males and females in our study is to be attributed, at least partially, to hormonal factors, it is worth noting that fasting unmasked the expected lower RER in females.

From a metabolic and substrate availability perspective, all these findings are very similar to those observed in endurance athletes practicing a ketogenic diet (KD). This diet is indeed an increasingly popular conditioning strategy during endurance training in athletes to activate fat metabolism pathways, increase utilization of fat as a primary muscle fuel while achieving very-low glycogen stores, and expose the body to circulating ketones<sup>37,38</sup>. While scientists keep exploring the benefits and drawbacks of KD
as an ergogenic strategy, several studies have explored its impact on endurance performance. After a 3-week KD, two studies³⁹,⁴⁰ showed significant increases in VO2max, while three others found only a preservation of VO2max⁴¹⁻⁴³. All studies reported a shift in the RER ratio, illustrating the greater reliance on fat oxidation in ketosis.

## MR Relaxometry, 1H muscle MRS, and tissue fat composition

In Skeletal muscle, PDFF measures, representing the overall lipid content, increased significantly during fasting and returned to baseline at M+1.

Skeletal muscle T2 relaxation times remained nearly stable at the end-fasting. This measure is known to be sensitive to both free water content and, to a lesser degree, lipid content, which can theoretically counteract each other⁴⁴⁻⁴⁶. T2*, on the other hand, increased markedly at end-fasting but returned to baseline values at M+1. T2* is notably influenced by the size of lipid droplets (LDs). In skeletal muscle and liver, smaller cytoplasmic LDs have longer T2* values than larger ones. T2* increases because smaller LDs have a higher surface-to-volume ratio, allowing more contact between lipid-associated and mobile water protons⁴⁷. The relatively greater surface area of smaller LDs also translates into an increased interface for lipid metabolic enzymes and potentially a greater capacity to mobilize lipids⁴⁸. Additionally, during energy stress conditions such as exercise, LD size can adapt and decrease within minutes⁴⁹. As lipids represent the primary energy substrate, stored TAGs in LD undergo increased delivery to mitochondria. The enhanced beta-oxidation rate and fatty acid trafficking lead to LD shrinking in tissues like muscle due to increased demand⁵⁰. During nutrient stress conditions such as during fasting, increased FA trafficking involves several mechanisms that are codependent on LD lipolysis, lipophagy, and mitochondrial fusion dynamics⁵¹,⁵². When increased accessibility of lipid fuels is needed, LD configuration is changing with more numerous but smaller LDs, helping a rapid supply of fat to mitochondria, while LDs are predominantly located very close or in direct contact with mitochondria⁵⁰,⁵³.

1H-MRS in muscles enables the localization and quantification of lipids in tissues and showed a very significant increase of the intracellular lipids (IMCL) at D+12, while the extracellular lipids pool (EMCL) lowered significantly. This redistribution of lipid in muscle favoring intracellular lipid
components occurs within the first days of fasting, as described after a 3-day⁵⁴ and 5-day fast⁵⁵. While none of these studies addressed the dynamics after food reintroduction, we showed that IMCL quickly returned to pre-fasting levels. At the same time, EMCL continued to fall after one month of fasting, with a muscle fat content as measured by PDFF below baseline values. This dynamic clearly illustrates the adaptive reciprocity of lipid and glucose metabolism, which switches from one system to another⁵⁶.

In both BMAT and SAT tissues, the proportions of PUFAs and MUFAs decreased and were mobilized during fasting. Mobilization of FAs has been demonstrated to be selective, depending on chain length, unsaturation, and isomerism⁵⁷,⁵⁸. The mobilization selectivity is linked to the dispersion of TAGs within lipid droplets depending on their polarity. The shorter and less saturated FAs, which are more polar, are positioned on the outer edge of the lipid droplet. This arrangement allows them to be more easily targeted by hormone-sensitive lipases, thereby safeguarding the longer-chain FAs⁵⁹.

Taurine is the most abundant free amino acid in skeletal muscle, implicated in critical cellular processes including substrates regulation (uptake, storage, and oxidation), the excitation-contraction processes, the cellular osmoregulation, and has antioxidant activity⁶⁰,⁶¹. Using 1H-MRS measures, we showed that long-term fasting led to conserving muscle taurine content. While acute conditions such as the exercise of various duration in rats may lead to decreased taurine concentration⁶², our findings agree with other experimental data showing the upregulation of taurine under starved conditions involving the cysteine and coenzyme-A pathways in taurine biosynthesis by the liver⁶³.

Choline is a metabolite that affects skeletal muscle by modulating fat and protein metabolism, inflammation, apoptosis, and autophagy⁶⁴,⁶⁵. While Choline endogenous synthesis is insufficient to support human choline requirements⁶⁶, we showed that 12-day fasting did not affect Choline skeletal muscle content.

## 31-P MRS and exercise muscle metabolism

Dynamic 31P-MRS enabled us to repeatedly and non-invasively measure variations in high-energy phosphorylated metabolites in a calibrated and sub-maximum exercise, thereby avoiding glycolytic metabolism. The kinetics of post-exercise recovery of PCr provided unique information regarding mitochondrial oxidative capacity²²,⁶⁷,⁶⁸. Overall, all time constants, τPCrRec (the rate of PCr resynthesis), ViPCr (maximal mitochondrial ATP production), and Vmax (maximal efficiency of
oxidative phosphorylation, ~ mitochondrial density) remained preserved after long-term fasting. More interesting is the finding that females improved their mitochondrial function significantly (lower τPCrRec, higher ViPCr, and Vmax) not only compared to males but also from their baseline value at the end of fasting. This improvement persisted one month after food reintroduction. This finding is coherent with the lower RER measured during cardiopulmonary exercise testing and sheds light on the greater capability of fat oxidation in females.

Several gender-related physiological differences explain the improved capabilities of fat oxidation in females⁶⁹. Females have a more significant proportion of slow-twitch fibers with greater vasodilatory capacity and capillarization⁷⁰. Estrogen has been shown to upregulate genes involved in fat metabolism, including lipoprotein lipase. Substrate utilization is improved due to greater mRNA expression of genes associated with fatty acid metabolism, including CD36⁷¹, and the presence of estrogen-mediated reserves of IMCL supports the fuel demands with a more significant percentage of IMCL in contact with mitochondria when compared to males⁷². In addition, Montero et al.⁷³ showed that improved fat oxidation during exercise is underpinned by augmented mitochondrial volume density and better fatty acid and lactate oxidative capacity of skeletal muscle. Earlier studies have been conducted during 2 to 5-day fasting experiments during near- or maximal exercises that lead to exercise-induced acidosis and lactate release⁷⁴⁻⁷⁶. A minimal pH lowering was observed at D+12 compared to baseline but did not impact exercise since pH remained stable at the end of the exercise. This minimal pH difference may be due to the balance in physiological ketosis between acetoacetate, acetone, and beta-hydroxybutyrate⁷⁷. The kinetics of Pi recovery (τPi) showed a significant difference, with a longer return to the basal state. Unlike the dynamics of PCr recovery, Pi recovery is not only due to mitochondrial respiration but is dependent on other factors, including pH buffering capacity⁷⁸.

## Conclusions

Our results indicate no disturbance of muscle metabolism caused by long-term fasting, with no evidence of a negative impact on mitochondrial respiration or general muscle cell function. The recorded muscle utilisation, although not negligible, has no impact on muscle strength and does not affect healthy human muscle performance in anaerobic lactic efforts. Our results also demonstrate an adaptation of the
mitochondrial respiration supply chain with lipid trafficking from the extracellular to intracellular compartment. This adaptation is coherent with the RER decrease, indicating a shift in substrate utilization, with a preference for lipids after the fasting period, showcasing the harmlessness of fasting for muscle metabolism. This preservation of muscle function during fasting suggests that humans, like wild animals, were evolutionary adapted by seasonal food shortages to fast periodically. This further suggests that there is no muscular contraindication in the use of this therapeutic protocol.

This adaptive phenomenon is likely geared towards ensuring better availability of fatty acids, which become the primary energetic substrate of the mitochondrial respiratory chain. Since the diverse intracellular compartments contribute to global cell homeostasis and the intracellular trafficking of lipids and proteins are closely related, these results open new perspectives while warranting further investigations. Of crucial interest is the improvement of the first ventilatory threshold, which, from a functional point of view, directly impacts everyday activities performed at a lower relative level of the maximal level, with immediate consequences on perceived fatigue.
Figures and Tables legends

**Fig 1** Global overview of the MRI and MRS protocol including: i) 31P dynamic MRS coupled to exercise using an MR ergometer, ii) 1H-MRS using SVS-STEAM MRS to investigate lipid composition and IMCL/EMCL sub-composition, iii) 1H high resolution 3D MRI for muscle volume quantification and CSE-MRI for advanced tissue characterization

**Fig 2** Illustration displaying A) the different steps and time-course of the lower leg exercise using the ergometer within the MR scanner (B); C) Typical 31P spectrum with highlighted main peaks of metabolites of interest, D) 3D overview of SRM-31P dynamic time acquisition; E) derived temporal curves of metabolites over time. F) Metabolites or quantities derived from modeling are listed below the graph from each protocol phase

**Fig 3** Upper and lower leg muscular volume measurements obtained before (D-1), at the end (D+12), and one month (M+1) after the end of fasting; left: global results, right: age and sex sub-group results

**Fig 4** Cardiopulmonary exercise test results obtained before (D-1), at the end (D+12), and one month (M+1) after the end of fasting, including oxygen uptake VO2 measures (top: maximum oxygen uptake capacity (VO2max), VO2 achieved at the first and second ventilatory threshold (VT1 and VT2), and the corresponding ergometric power levels. Data are mean±SD

**Fig 5** Respiratory exchange ratio (RER) during cardiopulmonary exercise tests, before (D-1), at the end (D+12), and one month (M+1) after the end of fasting. Data are mean±SD

**Fig 6-A:** Intra-myocellular lipids (IMCL) and extra-myocellular lipids (EMCL) content changes using 1H-MRS before (D-1), at the end (D+12), and one month (M+1) after the end of fasting; **-B:** Mitochondrial oxidative capacity estimates using 31P-MRS before (D-1), at the end (D+12), and one month (M+1) after the end of fasting. Data are mean±SD
**Table 1** Weight, upper-leg (UL) and lower-leg (LL) skeletal muscle volumes, UL and LL maximum voluntary contraction (MVC) force measurements (mean (SD), on overall, and stratified by sex and age). The table presents the effects of time, sex, and age, using a repeated-measures mixed-effects model. When applicable, pairwise post-hoc comparisons used a Tukey test to correct for multiple comparisons. For all analyses, significance was accepted at p< 0.05. (Legend for post-hoc analysis: D+12 & M+1 vs D-1 : *'=p<0.05; **'=p<0.01; ***'=p<0.001)

**Table 2:** Absolute (ml) and relative (%) skeletal muscle volume differences (right tight, right lower leg, right leg (tight+lower legs), and the extrapolated muscular volume changes on the combined right and left legs (assuming a right/left symmetry))
**Conflict of interest section:**

Antoine Naegel, Magalie Viallon, Helène Ratiney, Thu Nguyen, Benjamin Leporq, Djahid Kennouche, Thomas Grenier, Jean-Michel Guy and Pierre Croisille declare that they have no conflict of interest.

Franziska Grundler, Robin Mesnage and Françoise Wilhelmi de Toledo are employed by Buchinger
Wilhelmi Development & Holding GmbH in Überlingen.

Robin Schulze declares no conflict of interest.

**Funding**

This study was funded by the CHU de Saint-Etienne and Buchinger Wilhelmi Development & Holding GmbH, Überlingen, Germany. No additional external funding received for this study.

**Authors' contributions**

Pierre Croisille, Magalie Viallon, Robin Mesnage, Franziska Grundler and Françoise Wilhelmi de Toledo, conceived and conceptualized the study. Pierre Croisille, Magalie Viallon acquired the MR data and were responsible of the MR protocols. Franziska Grundler was responsible for the project administration and blood data collection. Antoine Naegel, Hélène Ratiney analyzed all the MRS data. Thu Nguyen, Thomas Grenier, Benjamin Leporq, Magalie Viallon joined efforts for the analysis of the MRI muscle data. Pierre Croisille, Antoine Naegel and Magalie Viallon realized and/or verified the statistical analyses of the data. All authors actively participated to the manuscript editing and reviewing.
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Acknowledgments

The authors thank Tom Hilbert from the Advanced Clinical Imaging Technology Siemens, (Lausanne, Switzerland) for the loan of the Model-based Accelerated T2 Mapping Work-in-Progress sequence (N4 VE11A_899C_Z002 SHUC MODEL BASED T2MAP). This work was partially supported by the LABEX PRIMES (ANR-11-LABX-0063). The 31P MR coils, Ergospect ergometer were funded by the FEDER program, Saint-Etienne Metropole (SME) and Conseil General de la Loire (CG42) and CPER.
We also thank the 32 participants of this study for their generous contribution.
<figure><img src="image_2.png" /></figure>

31P-MRS

<figure><img src="image_3.png" /></figure>

Surface coil
Tx/Rx ³¹P/¹H

3T MRI

<figure><img src="image_4.png" /></figure>

3D Dixon

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

Multi-echo CSE
Relaxometry
Body composition

<figure><img src="image_7.png" /></figure>

1H-MRS

1H-MRI
<figure><img src="image_8.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Lower right muscular volumes (global)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Upper right muscular volumes (sub-group analysis)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Upper right muscular volumes (sub-group analysis)</figcaption></figure>

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /></figure>
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>
<table><caption>Table 1</caption><thead><tr><th rowspan="2">Variables</th><th colspan="4">Time</th><th colspan="4">Sex</th><th colspan="4">Age</th><th colspan="4">Interactions</th></tr><tr><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th></tr></thead><tbody><tr><td>weight (kg)</td><td>78.8 (13.1)</td><td>72.9 (12.2)**</td><td>74.7 (12.2)**</td><td>0.002</td><td>male</td><td>85.2 (12.1)</td><td>78.4 (11.9)</td><td>80.6 (11.3)</td><td>0.12</td><td>young</td><td>80.0 (11.1)</td><td>74.0 (10.5)</td><td>74.5 (9.9)</td><td>0.934</td><td>0.051</td><td>0.072</td><td>0.729</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>female</td><td>72.8 (9.7)</td><td>67.7 (9.1)</td><td>68.8 (8.8)</td><td></td><td>old</td><td>77.7 (15.2)</td><td>71.9 (14.1)</td><td>74.9 (14.6)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>UL muscle volume (ml)</td><td>3617(965)</td><td>3403(890)**</td><td>3492(953)**</td><td>&lt;0.0001</td><td>male</td><td>4444(726)</td><td>4175(639)**</td><td>4299(712)**</td><td>&lt;0.0001</td><td>young</td><td>3786 (997)</td><td>3556(894)</td><td>3691(963)</td><td>0.068</td><td>0.051</td><td>0.078</td><td>0.618</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>female</td><td>2886(367)</td><td>2722(355)**</td><td>2797(383)**</td><td></td><td>old</td><td>3424(922)</td><td>3228(883)</td><td>3265(921)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>UL MVC (N.*m)</td><td>224.2 (76.6)</td><td>231.1 (81.0)</td><td>226.7 (80.0)</td><td>0.138</td><td>male</td><td>263.2 (57.4)</td><td>273.7 (66.8)</td><td>267.1 (65.4)</td><td>0.0003</td><td>young</td><td>249.4 (72.1)</td><td>254.9 (74.8)</td><td>245.4 (75.0)</td><td>0.011</td><td>0.530</td><td>0.078</td><td>0.198</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>female</td><td>187.5 (75.6)</td><td>191.0 (73.7)</td><td>186.3 (74.0)</td><td></td><td>old</td><td>197.4 (74.2)</td><td>205.8 (81.9)</td><td>207.9 (82.8)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LL muscle volume (ml)</td><td>1433(285)</td><td>1376(276)**</td><td>1390(279)**</td><td>&lt;0.0001</td><td>male</td><td>1643(254)</td><td>1567(266)**</td><td>1600(247)**</td><td>&lt;0.0001</td><td>young</td><td>1472(295)</td><td>1416(278)</td><td>1440(285)</td><td>0.229</td><td>0.007</td><td>0.166</td><td>0.420</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>female</td><td>1247(150)</td><td>1208(145)**</td><td>1205(140)**</td><td></td><td>old</td><td>1389(276)</td><td>1330(275)</td><td>1335(270)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LL MVC (N.*m)</td><td>680.1 (122.9)</td><td>712.4 (84.9)</td><td>706.8 (71.1)</td><td>0.089</td><td>male</td><td>725.7(78.9)</td><td>737.1(60.1)</td><td>739.5(37.9)</td><td>0.016</td><td>young</td><td>699.0(92.5)</td><td>723.7(83.2)</td><td>707.2(64.8)</td><td>0.403</td><td>0.405</td><td>0.394</td><td>0.420</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>female</td><td>637.1(142.5)</td><td>689.1(99.1)</td><td>676.0(81.7)</td><td></td><td>old</td><td>660.0(149.1)</td><td>700.3(87.7)</td><td>706.3(79.4)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>Table 2</caption><thead><tr><th></th><th>Group</th><th>Volume D-1 (ml)</th><th>Volume D+12 (ml)</th><th>Volume M+1 (ml)</th><th>Absolute Volume difference D+12 vs. D-1 (ml)</th><th>Absolute Volume difference M+1 vs. D-1 (ml)</th><th>Relative Volume difference D+12 vs. D-1 (%)</th><th>Relative Volume difference M+1 vs. D-1 (%)</th></tr></thead><tbody><tr><td rowspan="3">Right Thigh</td><td>all</td><td>3617</td><td>3403</td><td>3492</td><td>-214</td><td>-125</td><td>-5,9</td><td>-3,5</td></tr><tr><td>male</td><td>4444</td><td>4175</td><td>4299</td><td>-269</td><td>-145</td><td>-6,1</td><td>-3,3</td></tr><tr><td>female</td><td>2886</td><td>2722</td><td>2797</td><td>-164</td><td>-89</td><td>-5,7</td><td>-3,1</td></tr><tr><td rowspan="3">Right Lower Leg (LL)</td><td>all</td><td>1433</td><td>1376</td><td>1390</td><td>-57</td><td>-43</td><td>-4,0</td><td>-3,0</td></tr><tr><td>male</td><td>1643</td><td>1567</td><td>1600</td><td>-76</td><td>-33</td><td>-4,6</td><td>-2,6</td></tr><tr><td>female</td><td>1247</td><td>1208</td><td>1205</td><td>-39</td><td>3</td><td>-3,1</td><td>-3,4</td></tr><tr><td rowspan="3">Right Leg (Tigh+LL)</td><td>all</td><td>5050</td><td>4779</td><td>4882,0</td><td>-271</td><td>-168</td><td>-5,4</td><td>-3,3</td></tr><tr><td>male</td><td>6087</td><td>5742</td><td>5899,0</td><td>-345</td><td>-157</td><td>-5,7</td><td>-3,1</td></tr><tr><td>female</td><td>4133</td><td>3930</td><td>4002,0</td><td>-203</td><td>-72</td><td>-4,9</td><td>-3,2</td></tr><tr><td rowspan="3">Right and Left Legs</td><td>all</td><td>10100</td><td>9558</td><td>9764</td><td>-542</td><td>-336</td><td>-5,4</td><td>-3,3</td></tr><tr><td>male</td><td>12174</td><td>11484</td><td>11798</td><td>-690</td><td>-314</td><td>-5,7</td><td>-3,1</td></tr><tr><td>female</td><td>8266</td><td>7860</td><td>8004</td><td>-406</td><td>-144</td><td>-4,9</td><td>-3,2</td></tr></tbody></table>
<table><caption>Table S1</caption><thead><tr><th>Parameters</th><th>CSE-MRI</th><th>Dixon-3D-GRE</th></tr></thead><tbody><tr><td>Sequence Type</td><td>3D Multi-echo-gradient-echo</td><td>3D Multi-echo-gradient-echo</td></tr><tr><td>Number of echoes</td><td>10</td><td>2</td></tr><tr><td>Acquisition Plane</td><td>Transversal</td><td>Coronal</td></tr><tr><td>FOV</td><td>400*250</td><td>500*312</td></tr><tr><td>Slice Thickness (mm)</td><td>5</td><td>1</td></tr><tr><td>Acq. Pixel size (mm²)</td><td>1.3 * 1.3 * 2</td><td>1.2 * 1.2 * 1.2</td></tr><tr><td>Matrix size</td><td>160*128</td><td>260*416</td></tr><tr><td>Repetition time (msec)</td><td>15.50</td><td>4.08</td></tr><tr><td>Echo time (msec)</td><td>12 echos 1..2/14.40</td><td>1.29/2.52</td></tr><tr><td>Flip Angle(°)</td><td>5°</td><td>9°</td></tr><tr><td>Bandwidth(Hz/Px)</td><td>1560</td><td>1000</td></tr><tr><td>Averages</td><td>1</td><td>1</td></tr><tr><td>Fat suppression</td><td>None</td><td>Dixon</td></tr><tr><td>Partial Fourier</td><td>7/8</td><td>7/8</td></tr><tr><td>Total Acq. Time (min)</td><td>2:08</td><td>5:16</td></tr></tbody></table>

<table><thead><tr><th>Parameters</th><th>1H-MRS</th><th>31P-MRS</th></tr></thead><tbody><tr><td>Sequence Type</td><td>Single Voxel STEAM</td><td>FID</td></tr><tr><td>Number of samples</td><td>2048</td><td>512</td></tr><tr><td>Acq. Pixel size (mm²)</td><td>30*30*30</td><td>-</td></tr><tr><td>Voxel size (mm³)</td><td>27000</td><td>-</td></tr><tr><td>Nex/ Averages</td><td>180</td><td>-</td></tr><tr><td>Repetition time (msec)</td><td>2000</td><td>4</td></tr><tr><td>Echo time (msec)</td><td>20</td><td>0.35</td></tr><tr><td>Flip Angle(°)</td><td>90</td><td>90</td></tr><tr><td>Bandwidth(Hz)</td><td>3000</td><td>2500</td></tr><tr><td>Water suppression</td><td>yes</td><td>none</td></tr></tbody></table>
<figure><img src="image_26.png" /></figure>
<table><caption>Table S2</caption><thead><tr><th>sex</th><th>age group</th><th>N subjects</th><th>age (years)</th><th>height (cm)</th><th>Weight (kg)</th><th>BMI (kg/m2)</th></tr></thead><tbody><tr><td>Female</td><td>young (&lt;50)</td><td>8</td><td>38 (7.45)</td><td>168.33 (5.2)</td><td>71.88 (9.65)</td><td>25.41 (3.59)</td></tr><tr><td>Female</td><td>aged (≥50)</td><td>8</td><td>60.25 (7.01)</td><td>165.88 (5.62)</td><td>73.8 (10.33)</td><td>26.96 (4.65)</td></tr><tr><td>Male</td><td>young (&lt;50)</td><td>8</td><td>38.25 (5.47)</td><td>182.88 (5.3)</td><td>84.33 (17.16)</td><td>25.12 (4.52)</td></tr><tr><td>Male</td><td>aged (≥50)</td><td>8</td><td>57.13 (4.94)</td><td>178.88 (6.06)</td><td>86.15 (8.47)</td><td>26.98 (3.04)</td></tr><tr><td>Total</td><td></td><td>32</td><td>48.09 (12.11)</td><td>173.82 (8.89)</td><td>78.82 (12.95)</td><td>26.1 (3.9)</td></tr></tbody></table>
<table><caption>Table S3</caption><thead><tr><th>Activity level category</th><th>D-1</th><th>D+12</th><th>M+1</th></tr></thead><tbody><tr><td>Vigorous (min)</td><td>168 (56.31)</td><td>158.73 (41.66)</td><td>171.79 (52.82)</td></tr><tr><td>Moderate (min)</td><td>153.63 (30.19)</td><td>132.32 (18.87)</td><td>164.17 (22.66)</td></tr><tr><td>Moderate-to-Vigorous (min)</td><td>321.62 (72.37)</td><td>291.05 (44.88)</td><td>335.97 (58.2)</td></tr><tr><td>Low (min)</td><td>240.28 (46.32)</td><td>209.74 (40.92)</td><td>250.49 (46.84)</td></tr><tr><td>Sedentary (min)</td><td>384.02 (67.12)</td><td>466.02 (46.79)</td><td>409.16 (93.5)</td></tr></tbody></table>
<table><caption>Table S4</caption><thead><tr><th rowspan="2">Variables</th><th colspan="4">Time</th><th colspan="4">Sex</th><th colspan="4">Age</th><th colspan="4">Interactions</th></tr><tr><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>Time*Sex</th><th>Time*Age</th><th>Sex*Age</th></tr></thead><tbody><tr><td>VO<sub>2</sub>max (ml/min/kg)</td><td>31.4 (10.0)</td><td>32.6 (9.1)</td><td>31.1 (6.3)</td><td>0.669</td><td>male<br>female</td><td>35.1 (9.3)<br>27.2 (9.3)*</td><td>35.9 (9.2)<br>28.5 (7.6)*</td><td>34.3 (4.9)<br>26.9 (5.4)**</td><td>0.010</td><td>young<br>old</td><td>34.7 (11.2)<br>28.1 (7.6)</td><td>35.5 (9.1)<br>29.5 (8.4)</td><td>32.0 (6.1)<br>30.4 (6.5)</td><td>0.050</td><td>0.561</td><td>0.168</td><td>0.701</td></tr><tr><td>VO<sub>2</sub>@VT1 (ml/min/kg)</td><td>19.0 (6.9)</td><td>21.2 (6.4)</td><td>19.8 (4.5)</td><td>0.099</td><td>male<br>female</td><td>20.8 (6.9)<br>16.9 (6.5)</td><td>22.0 (6.9)<br>20.2 (5.8)</td><td>22.5 (3.7)<br>16.1 (2.5)</td><td>0.041</td><td>young<br>old</td><td>20.0 (8.0)<br>17.9 (5.4)</td><td>23.0 (6.7)<br>19.3 (5.7)</td><td>18.6 (3.5)<br>20.7 (5.2)</td><td>0.351</td><td>0.258</td><td>0.0257</td><td>0.295</td></tr><tr><td>VO<sub>2</sub>@VT2 (ml/min/kg)</td><td>25.7 (8.9)</td><td>27.7 (7.4)</td><td>28.3 (5.9)</td><td>0.091</td><td>male<br>female</td><td>28.7 (8.4)<br>22.3 (8.5)</td><td>30.5 (7.3)<br>24.0 (6.0)*</td><td>31.7 (4.5)<br>23.7 (4.2)**</td><td>0.002</td><td>young<br>old</td><td>28.7 (10.2)<br>22.8 (6.5)</td><td>30.3 (6.7)<br>24.6 (7.1)</td><td>28.4 (5.1)<br>28.2 (6.7)</td><td>0.031</td><td>0.999</td><td>0.042</td><td>0.344</td></tr><tr><td>Pmax @VO<sub>2</sub>max (W)</td><td>200.9 (63.5)</td><td>195.4 (62.5)</td><td>219.6 (56.4)**</td><td>&lt;0.001</td><td>male<br>female</td><td>242.8 (50.8)<br>153.0 (37.3)***</td><td>238.4 (43.7)<br>142.6 (35.1)***</td><td>256.2 (40.3)<br>169.6 (30.4)***</td><td>&lt;0.001</td><td>young<br>old</td><td>213.1 (62.5)<br>188.7 (64.3)</td><td>201.4 (59.4)<br>189.1 (67.2)</td><td>220.5 (56.0)<br>218.7 (59.1)</td><td>0.255</td><td>0.296</td><td>0.150</td><td>0.405</td></tr><tr><td>P @VT1 (W)</td><td>116.7 (42.8)</td><td>115.4 (33.5)</td><td>122.9 (37.8)</td><td>0.206</td><td>male<br>female</td><td>136.0 (40.7)<br>92.8 (33.0)**</td><td>132.8 (29.2)<br>94.0 (25.4)**</td><td>147.8 (25.9)<br>89.8 (22.4)**</td><td>&lt;0.001</td><td>young<br>old</td><td>117.7 (39.8)<br>115.5 (47.3)</td><td>121.8 (32.5)<br>108.6 (34.3)</td><td>116.5 (34.6)<br>128.4 (40.7)</td><td>0.676</td><td>0.394</td><td>0.024</td><td>0.074</td></tr><tr><td>P @VT2 (W)</td><td>162.0 (52.4)</td><td>161.9 (48.7)</td><td>13.4 (115.7)**</td><td>&lt;0.001</td><td>male<br>female</td><td>194.8 (42.1)<br>121.8 (31.8)***</td><td>194.9 (31.9)<br>117.9 (27.1)***</td><td>226.9 (29.1)<br>195.3 (176.4)***</td><td>&lt;0.001</td><td>young<br>old</td><td>170.5 (51.3)<br>152.9 (53.8)</td><td>169.3 (44.5)<br>153.3 (53.6)</td><td>243.5 (156.9)<br>187.2 (56.2)</td><td>0.081</td><td>0.873</td><td>0.495</td><td>0.134</td></tr><tr><td>RER</td><td>1.10 (0.09)</td><td>1.00 (0.08)***</td><td>1.29 (0.13)***</td><td>&lt;0.001</td><td>male<br>female</td><td>1.10 (0.08)</td><td>0.96 (0.08)</td><td>1.29 (0.10)</td><td>0.490</td><td>young<br>old</td><td>1.10 (0.09)<br>1.09 (0.09)</td><td>1.00 (0.08)<br>1.01 (0.09)</td><td>1.28 (0.10)<br>1.28 (0.10)</td><td>0.957</td><td>0.017</td><td>0.827</td><td>0.349</td></tr></tbody></table>
<table><caption>Table S5</caption><thead><tr><th rowspan="2">Variables</th><th colspan="4">Time</th><th colspan="4">Sex</th><th colspan="4">Age</th><th colspan="4">Interactions</th></tr><tr><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+10</th><th>p</th><th>p</th><th>p</th><th>p</th><th>p</th></tr></thead><tbody><tr><td rowspan="4">Skeletal Muscle</td><td rowspan="2">T2 (ms)</td><td rowspan="2">41,46 (5,5)</td><td rowspan="2">42,54 (4,6)</td><td rowspan="2">41,79 (4,8)</td><td rowspan="2">0.344</td><td>male</td><td>40,07 (6,9)</td><td>40,99 (6,3)</td><td>40,50 (6,1)</td><td rowspan="2">0.228</td><td>young</td><td>40,48 (5,3)</td><td>41,95 (4,9)</td><td>40,27 (4,5)</td><td rowspan="2">0.140</td><td rowspan="2">0.954</td><td rowspan="2">0.510</td><td rowspan="2">0.622</td></tr><tr><td>female</td><td>42,50 (4,10)</td><td>43,72 (2,3)</td><td>42,86 (3,4)</td><td>old</td><td>42,77 (5,8)</td><td>43,30 (4,2)</td><td>43,64 (4,7)</td></tr><tr><td rowspan="2">T2* (ms)</td><td rowspan="2">24,2 (0,9)</td><td rowspan="2">25,4 (1,0)***</td><td rowspan="2">24,4 (1,0)*</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>24,1 (0,8)</td><td>25,2 (1,1)</td><td>24,3 (0,9)</td><td rowspan="2">0.350</td><td>young</td><td>24,5 (0,5)</td><td>25,8 (0,9)</td><td>24,9 (0,8)</td><td rowspan="2">0.0145</td><td rowspan="2">0.506</td><td rowspan="2">0.831</td><td rowspan="2">0.434</td></tr><tr><td>female</td><td>24,2 (1,0)</td><td>25,6 (0,9)</td><td>24,6 (1,1)</td><td>old</td><td>23,8 (1,0)*</td><td>25,0 (1,0)*</td><td>24,0 (1,1)*</td></tr><tr><td rowspan="2">PDFF (%)</td><td rowspan="2">8,7 (2,2)</td><td rowspan="2">8,9 (2,1)*</td><td rowspan="2">8,5 (2,0)</td><td rowspan="2">0.002</td><td>male</td><td>8,2 (2,3)</td><td>8,2 (2,2)</td><td>8,0 (2,1)</td><td rowspan="2">0.115</td><td>young</td><td>7,8 (1,5)</td><td>8,0 (1,3)</td><td>7,7 (1,6)</td><td rowspan="2">0.019</td><td rowspan="2">0.314</td><td rowspan="2">0.214</td><td rowspan="2">0.436</td></tr><tr><td>female</td><td>9,2 (2,1)</td><td>9,5 (1,8)</td><td>9,0 (1,9)</td><td>old</td><td>9,5 (2,5)*</td><td>9,8 (2,4)*</td><td>9,2 (2,2)*</td></tr><tr><td rowspan="4">1H-MRS Muscle</td><td rowspan="2">IMCL/Cr</td><td rowspan="2">4,44 (2,80)</td><td rowspan="2">9,58 (5,64)*</td><td rowspan="2">4,97 (3,03)</td><td rowspan="2">0.009</td><td>male</td><td>5,22 (3,78)</td><td>11,00 (6,48)</td><td>6,28 (3,15)</td><td rowspan="2">0.038</td><td>young</td><td>3,56 (1,43)</td><td>8,70 (5,24)</td><td>4,65 (2,73)</td><td rowspan="2">0.207</td><td rowspan="2">0.842</td><td rowspan="2">0.912</td><td rowspan="2">0.469</td></tr><tr><td>female</td><td>3,66 (1,08)</td><td>7,92 (4,44)</td><td>3,81 (2,55)</td><td>old</td><td>5,58 (3,77)</td><td>10,99 (6,58)</td><td>5,34 (3,50)</td></tr><tr><td rowspan="2">EMCL/Cr</td><td rowspan="2">8,30 (5,99)</td><td rowspan="2">5,93 (3,38)*</td><td rowspan="2">5,38 (5,73)**</td><td rowspan="2">0.029</td><td>male</td><td>8,58 (4,68)</td><td>6,32 (4,20)</td><td>5,43 (4,62)</td><td rowspan="2">0.954</td><td>young</td><td>7,33 (5,24)</td><td>6,64 (3,65)</td><td>4,42 (4,91)</td><td rowspan="2">0.758</td><td rowspan="2">0.988</td><td rowspan="2">0.237</td><td rowspan="2">0.958</td></tr><tr><td>female</td><td>8,09 (7,12)</td><td>5,53 (2,68)</td><td>5,33 (6,75)</td><td>old</td><td>9,55 (7,06)</td><td>4,92 (3,05)</td><td>6,60 (6,85)</td></tr><tr><td rowspan="4">Cho/Cr</td><td rowspan="2">0,84 (0,36)</td><td rowspan="2">0,92 (0,34)</td><td rowspan="2">0,78 (0,27)</td><td rowspan="2">0.183</td><td>male</td><td>1,04 (0,33)</td><td>1,12 (0,33)</td><td>0,94 (0,28)</td><td rowspan="2">0.010</td><td>young</td><td>0,84 (0,33)</td><td>0,91 (0,39)</td><td>0,73 (0,13)</td><td rowspan="2">0.970</td><td rowspan="2">0.726</td><td rowspan="2">0.659</td><td rowspan="2">0.895</td></tr><tr><td>female</td><td>0,66 (0,29)</td><td>0,68 (0,14)</td><td>0,64 (0,15)</td><td>old</td><td>0,83 (0,40)</td><td>0,94 (0,27)</td><td>0,84 (0,37)</td></tr><tr><td rowspan="2">Tau / Cr</td><td rowspan="2">0,42 (0,21)</td><td rowspan="2">0,41 (0,18)</td><td rowspan="2">0,29 (0,16)</td><td rowspan="2">0.094</td><td>male</td><td>0,46 (0,23)</td><td>0,47 (0,20)</td><td>0,27 (0,19)</td><td rowspan="2">0.586</td><td>young</td><td>0,49 (0,25)</td><td>0,35 (0,14)</td><td>0,34 (0,17)</td><td rowspan="2">0.463</td><td rowspan="2">0.289</td><td rowspan="2">0.154</td><td rowspan="2">0.829</td></tr><tr><td>female</td><td>0,40 (0,20)</td><td>0,33 (0,13)</td><td>0,31 (0,14)</td><td>old</td><td>0,35 (0,12)</td><td>0,49 (0,22)</td><td>0,24 (0,15)</td></tr><tr><td rowspan="2">Cr / Water</td><td rowspan="2">0,12 (0,03)</td><td rowspan="2">0,12 (0,03)</td><td rowspan="2">0,14 (0,03)</td><td rowspan="2">0.230</td><td>male</td><td>0,11 (0,02)</td><td>0,10 (0,02)</td><td>0,13 (0,03)</td><td rowspan="2">0.207</td><td>young</td><td>0,12 (0,02)</td><td>0,12 (0,03)</td><td>0,15 (0,03)</td><td rowspan="2">0.203</td><td rowspan="2">0.222</td><td rowspan="2">0.210</td><td rowspan="2">0.191</td></tr><tr><td>female</td><td>0,13 (0,02)</td><td>0,14 (0,02)</td><td>0,16 (0,02)</td><td>old</td><td>0,12 (0,03)</td><td>0,12 (0,03)</td><td>0,14 (0,03)</td></tr><tr><td rowspan="4">SAT</td><td rowspan="2">T2* (ms)</td><td rowspan="2">26,7 (2,2)</td><td rowspan="2">26,2 (2,3)*</td><td rowspan="2">25,3 (2,3)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>25,3 (1,9)</td><td>24,6 (2,1)</td><td>23,7 (1,8)</td><td rowspan="2">&lt;0.0001</td><td>young</td><td>26,6 (1,9)</td><td>26,1 (2,5)</td><td>25,1 (2,3)</td><td rowspan="2">0.635</td><td rowspan="2">0.264</td><td rowspan="2">0.672</td><td rowspan="2">0.713</td></tr><tr><td>female</td><td>28,1 (1,5)***</td><td>27,8 (1,2)***</td><td>27,0 (1,2)***</td><td>old</td><td>26,8 (2,6)</td><td>26,4 (2,2)</td><td>25,5 (2,3)</td></tr><tr><td rowspan="2">PDFF (%)</td><td rowspan="2">90,0 (2,8)</td><td rowspan="2">89,8 (3,2)</td><td rowspan="2">88,6 (3,4)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>87,9 (2,3)</td><td>87,4 (2,7)</td><td>86,0 (2,7)</td><td rowspan="2">&lt;0.0001</td><td>young</td><td>90,0 (2,6)</td><td>89,6 (3,6)</td><td>88,4 (3,6)</td><td rowspan="2">0.685</td><td rowspan="2">0.005</td><td rowspan="2">0.112</td><td rowspan="2">0.631</td></tr><tr><td>female</td><td>92,1 (1,2)***</td><td>92,2 (1,3)***</td><td>91,3 (1,3)***</td><td>old</td><td>89,9 (3,0)</td><td>90,1 (2,9)</td><td>88,9 (3,2)</td></tr><tr><td rowspan="2">SFA (%)</td><td rowspan="2">58,0 (4,3)</td><td rowspan="2">58,9 (4,3)***</td><td rowspan="2">59,6 (4,6)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>61,1 (2,6)</td><td>62,1 (2,7)</td><td>62,9 (2,7)</td><td rowspan="2">&lt;0.0001</td><td>young</td><td>58,2 (3,9)</td><td>59,1 (4,4)</td><td>59,9 (4,4)</td><td rowspan="2">0.627</td><td rowspan="2">0.356</td><td rowspan="2">0.890</td><td rowspan="2">0.550</td></tr><tr><td>female</td><td>54,9 (3,4)***</td><td>55,7 (3,0)***</td><td>56,3 (3,5)***</td><td>old</td><td>57,8 (4,8)</td><td>58,6 (4,3)</td><td>59,3 (4,9)</td></tr><tr><td rowspan="2">MUFA (%)</td><td rowspan="2">34,0 (2,8)</td><td rowspan="2">33,4 (3,0)***</td><td rowspan="2">32,9 (3,1)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>31,9 (1,8)</td><td>31,1 (1,9)</td><td>30,5 (1,9)</td><td rowspan="2">&lt;0.0001</td><td>young</td><td>33,9 (2,6)</td><td>33,2 (3,2)</td><td>32,7 (3,2)</td><td rowspan="2">0.667</td><td rowspan="2">0.030</td><td rowspan="2">0.272</td><td rowspan="2">0.431</td></tr><tr><td>female</td><td>36,0 (1,8)***</td><td>35,7 (1,8)***</td><td>35,3 (2,1)***</td><td>old</td><td>34,0 (3,0)</td><td>33,6 (2,9)</td><td>33,1 (3,2)</td></tr><tr><td rowspan="2">PUFA (%)</td><td rowspan="2">8,0 (1,7)</td><td rowspan="2">7,6 (1,5)**</td><td rowspan="2">7,5 (1,6)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>7,0 (1,0)</td><td>6,7 (1,1)</td><td>6,5 (1,1)</td><td rowspan="2">&lt;0.0001</td><td>young</td><td>7,9 (1,5)</td><td>7,6 (1,5)</td><td>7,3 (1,5)</td><td rowspan="2">0.605</td><td rowspan="2">0.011</td><td rowspan="2">0.857</td><td rowspan="2">0.577</td></tr><tr><td>female</td><td>9,0 (1,7)***</td><td>8,6 (1,3)***</td><td>8,4 (1,5)***</td><td>old</td><td>8,2 (1,9)</td><td>7,7 (1,5)</td><td>7,6 (1,8)</td></tr><tr><td rowspan="4">Volume (cm3)</td><td rowspan="2">3187,2 (1630,3)</td><td rowspan="2">2770,0 (1374,4)**</td><td rowspan="2">2621,2 (1274,5)***</td><td rowspan="2">&lt;0.0001</td><td>male</td><td>2244,7 (1103,3)</td><td>1946,8 (1006,3)</td><td>1823,2 (889,5)</td><td rowspan="2">0.0002</td><td>young</td><td>3012,1 (1301,4)</td><td>2600,5 (1217,0)</td><td>2476,7 (1083,2)</td><td rowspan="2">0.423</td><td rowspan="2">0.167</td><td rowspan="2">0.921</td><td rowspan="2">0.310</td></tr><tr><td>female</td><td>4129,6 (1543,0)***</td><td>3593,3 (1202,3)***</td><td>3419,2 (1098,8)***</td><td>old</td><td>3362,2 (1932,3)</td><td>2939,6 (1536,8)</td><td>2765,6 (1462,6)</td></tr><tr><td rowspan="2">BMAT</td><td rowspan="2">T2* (ms)</td><td rowspan="2">17,8 (2,7)</td><td rowspan="2">17,3 (2,1)</td><td rowspan="2">17,6 (2,2)</td><td rowspan="2">0.130</td><td>male</td><td>18,9 (2,0)</td><td>17,8 (1,5)</td><td>18,2 (1,6)</td><td rowspan="2">0.082</td><td>young</td><td>18,2 (2,6)</td><td>17,3 (2,0)</td><td>17,8 (2,3)</td><td rowspan="2">0.641</td><td rowspan="2">0.043</td><td rowspan="2">0.183</td><td rowspan="2">0.826</td></tr><tr><td>female</td><td>16,8 (3,0)</td><td>16,8 (2,5)</td><td>17,1 (2,5)</td><td>old</td><td>17,4 (2,9)</td><td>17,4 (2,2)</td><td>17,4 (2,0)</td></tr><tr><td rowspan="4">BMAT</td><td rowspan="2">PDFF (%)</td><td rowspan="2">88,2 (7,0)</td><td rowspan="2">88,0 (5,2)</td><td rowspan="2">89,0 (4,4)</td><td rowspan="2">0.239</td><td>male</td><td>91,3 (2,6)</td><td>90,0 (2,9)</td><td>90,9 (2,2)</td><td rowspan="2">0.012</td><td>young</td><td>88,9 (4,5)</td><td>87,6 (3,9)</td><td>89,1 (3,4)</td><td rowspan="2">0.903</td><td rowspan="2">0.106</td><td rowspan="2">0.230</td><td rowspan="2">0.367</td></tr><tr><td>female</td><td>85,1 (8,7)</td><td>86,1 (6,3)</td><td>87,2 (5,3)</td><td>old</td><td>87,5 (9,0)</td><td>88,4 (6,4)</td><td>89,0 (5,4)</td></tr><tr><td rowspan="2">SFA (%)</td><td rowspan="2">60,0 (4,9)</td><td rowspan="2">60,6 (4,1)</td><td rowspan="2">59,4 (3,8)</td><td rowspan="2">0.022</td><td>male</td><td>59,0 (3,8)</td><td>60,3 (3,8)</td><td>59,0 (3,7)</td><td rowspan="2">0.457</td><td>young</td><td>59,9 (4,2)</td><td>60,8 (3,7)</td><td>59,2 (3,7)</td><td rowspan="2">0.949</td><td rowspan="2">0.097</td><td rowspan="2">0.557</td><td rowspan="2">0.133</td></tr><tr><td>female</td><td>61,0 (5,7)</td><td>60,8 (4,5)</td><td>59,8 (4,0)</td><td>old</td><td>60,2 (5,6)</td><td>60,4 (4,6)</td><td>59,6 (4,0)</td></tr><tr><td rowspan="2">MUFA (%)</td><td rowspan="2">32,3 (2,8)</td><td rowspan="2">32,1 (2,4)</td><td rowspan="2">32,8 (2,2)</td><td rowspan="2">0.026</td><td>male</td><td>33,1 (2,0)</td><td>32,4 (2,2)</td><td>33,1 (2,1)</td><td rowspan="2">0.278</td><td>young</td><td>32,4 (2,3)</td><td>32,0 (2,1)</td><td>32,7 (2,0)</td><td rowspan="2">0.949</td><td rowspan="2">0.072</td><td rowspan="2">0.597</td><td rowspan="2">0.182</td></tr><tr><td>female</td><td>31,6 (3,4)</td><td>31,8 (2,7)</td><td>32,4 (2,3)</td><td>old</td><td>32,3 (3,3)</td><td>32,3 (2,8)</td><td>32,8 (2,4)</td></tr><tr><td rowspan="2">PUFA (%)</td><td rowspan="2">7,7 (2,1)</td><td rowspan="2">7,3 (1,8)</td><td rowspan="2">7,8 (1,7)</td><td rowspan="2">0.0120</td><td>male</td><td>7,9 (1,8)</td><td>7,3 (1,7)</td><td>7,9 (1,7)</td><td rowspan="2">0.796</td><td>young</td><td>7,8 (2,0)</td><td>7,2 (1,7)</td><td>8,0 (1,8)</td><td rowspan="2">0.831</td><td rowspan="2">0.199</td><td rowspan="2">0.383</td><td rowspan="2">0.010</td></tr><tr><td>female</td><td>7,4 (2,4)</td><td>7,3 (1,8)</td><td>7,8 (1,8)</td><td>old</td><td>7,6 (2,3)</td><td>7,4 (1,9)</td><td>7,7 (1,7)</td></tr></tbody></table>
<table><caption>Table S6</caption><thead><tr><th rowspan="2">Variables</th><th colspan="4">Time</th><th colspan="4">Sex</th><th colspan="4">Age</th><th colspan="4">Interactions</th></tr><tr><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th><th>D-1</th><th>D+12</th><th>M+1</th><th>p</th></tr></thead><tbody><tr><td>[ADP] (μM)</td><td>6.66 (0.33)</td><td>6.37 (0.54)**</td><td>6.65 (0.33)</td><td>0.0022</td><td>male<br>6.82 (0.32)</td><td>6.47 (0.57)</td><td>6.76 (0.37)</td><td>0.037</td><td>young<br>6.63 (0.30)</td><td>6.27 (0.52)</td><td>6.75 (0.27)</td><td>0.886</td><td>old<br>6.69 (0.36)</td><td>6.48 (0.56)</td><td>6.54 (0.36)</td><td>0.041</td><td>0.573</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>[PCr] (mM)</td><td>33.91 (3.10)</td><td>33.21 (3.41)</td><td>32.92 (3.27)</td><td>0.402</td><td>male<br>6.51 (0.26)**</td><td>6.27 (0.52)</td><td>6.54 (0.24)*</td><td></td><td>young<br>6.69 (0.30)</td><td>6.27 (0.52)</td><td>6.75 (0.27)</td><td>0.886</td><td>old<br>6.69 (0.36)</td><td>6.48 (0.56)</td><td>6.54 (0.36)</td><td>0.041</td><td>0.573</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH rest</td><td>6.88 (0.02)</td><td>6.86 (0.03)**</td><td>6.88 (0.02)</td><td>&lt;0.0001</td><td>male<br>6.89 (0.02)</td><td>6.86 (0.03)</td><td>6.89 (0.02)</td><td>0.0156</td><td>young<br>6.88 (0.02)</td><td>6.85 (0.03)</td><td>6.89 (0.02)</td><td>0.972</td><td>old<br>6.88 (0.02)</td><td>6.86 (0.03)</td><td>6.87 (0.02)</td><td>0.035</td><td>0.188</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>[PI] (mM)</td><td>4.04 (0.66)</td><td>3.87 (0.67)</td><td>4.13 (0.84)</td><td>0.245</td><td>male<br>4.11 (0.57)</td><td>3.79 (0.62)</td><td>4.39 (0.92)</td><td>0.466</td><td>young<br>3.82 (0.65)</td><td>3.63 (0.72)</td><td>3.99 (0.91)</td><td>0.049</td><td>old<br>4.27 (0.60)</td><td>4.13 (0.53)*</td><td>4.28 (0.78)</td><td>0.107</td><td>0.751</td><td>0.468</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCr/PI</td><td>8.55 (1.18)</td><td>8.78 (1.44)</td><td>8.21 (1.33)</td><td>0.090</td><td>male<br>8.15 (1.04)</td><td>8.79 (1.67)</td><td>7.71 (1.34)</td><td>0.157</td><td>young<br>8.83 (1.36)</td><td>9.23 (1.58)</td><td>8.47 (1.28)</td><td>0.087</td><td>old<br>8.25 (0.91)</td><td>8.29 (1.13)</td><td>7.93 (1.37)</td><td>0.134</td><td>0.613</td><td>0.808</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>tPCrEx (s)</td><td>30.48 (15.93)</td><td>32.94 (13.53)</td><td>28.71 (8.25)</td><td>0.289</td><td>male<br>29.69 (16.27)</td><td>33.02 (10.08)</td><td>29.29 (8.06)</td><td>0.902</td><td>young<br>30.91 (12.82)</td><td>33.76 (11.61)</td><td>29.04 (8.23)</td><td>0.904</td><td>old<br>29.98 (19.54)</td><td>31.98 (15.97)</td><td>28.32 (8.61)</td><td>0.997</td><td>0.076</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>tPIEx (s)</td><td>25.19 (7.90)</td><td>25.19 (7.90)</td><td>23.10 (5.52)</td><td>0.209</td><td>male<br>23.71 (5.22)</td><td>23.71 (5.22)</td><td>22.35 (3.08)</td><td>0.363</td><td>young<br>26.78 (9.25)</td><td>26.78 (9.25)</td><td>23.94 (6.63)</td><td>0.261</td><td>old<br>23.95 (5.82)</td><td>23.95 (5.82)*</td><td>22.12 (3.93)</td><td>0.135</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>[PCr]Post (mM)</td><td>19.85 (3.89)</td><td>19.66 (3.39)</td><td>18.91 (3.55)</td><td>0.479</td><td>male<br>19.83 (3.26)</td><td>19.94 (2.94)</td><td>19.95 (3.23)</td><td>0.534</td><td>young<br>19.37 (3.84)</td><td>19.28 (3.28)</td><td>19.17 (3.79)</td><td>0.738</td><td>old<br>29.40 (4.04)</td><td>20.10 (3.59)</td><td>18.60 (3.38)</td><td>0.396</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>[PI]Post (mM)</td><td>13.93 (2.70)</td><td>14.22 (2.41)</td><td>15.08 (2.58)</td><td>0.055</td><td>male<br>12.65 (1.96)</td><td>13.15 (1.10)</td><td>14.53 (1.85)</td><td>0.029</td><td>young<br>13.34 (2.05)</td><td>13.72 (2.24)</td><td>15.24 (2.94)</td><td>0.543</td><td>old<br>14.62 (3.26)</td><td>14.80 (2.57)</td><td>14.89 (2.21)</td><td>0.482</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH Post</td><td>6.89 (0.03)</td><td>6.87 (0.03)**</td><td>6.89 (0.03)</td><td>0.0016</td><td>male<br>6.89 (0.02)</td><td>6.86 (0.02)</td><td>6.89 (0.03)</td><td>0.960</td><td>young<br>6.89 (0.03)</td><td>6.87 (0.03)</td><td>6.90 (0.02)</td><td>0.310</td><td>old<br>6.88 (0.02)</td><td>6.87 (0.03)</td><td>6.88 (0.03)</td><td>0.081</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCr</td><td>41.10 (11.33)</td><td>40.49 (9.27)</td><td>42.37 (10.27)</td><td>0.577</td><td>male<br>39.08 (10.68)</td><td>38.55 (7.73)</td><td>38.89 (8.78)</td><td>0.238</td><td>young<br>41.06 (11.30)</td><td>40.19 (8.54)</td><td>41.88 (9.64)</td><td>0.953</td><td>old<br>41.15 (11.87)</td><td>40.85 (10.43)</td><td>42.94 (11.36)</td><td>0.997</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Hydrolisis (%)</td><td>41.10 (11.33)</td><td>40.49 (9.27)</td><td>42.37 (10.27)</td><td>0.577</td><td>male<br>39.08 (10.68)</td><td>42.16 (10.40)</td><td>45.36 (10.80)</td><td>0.238</td><td>young<br>37.69 (7.33)</td><td>35.85 (9.42)</td><td>34.95 (7.89)</td><td>0.860</td><td>old<br>31.88 (7.86)</td><td>32.34 (8.28)</td><td>30.51 (5.56)</td><td>0.585</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ViPCr (mM/s)</td><td>0.41 (0.12)</td><td>0.41 (0.16)</td><td>0.44 (0.14)</td><td>0.457</td><td>male<br>0.36 (0.07)</td><td>0.32 (0.08)</td><td>0.35 (0.07)</td><td>0.0003</td><td>young<br>0.37 (0.07)</td><td>0.38 (0.12)</td><td>0.41 (0.12)</td><td>0.075</td><td>old<br>0.45 (0.15)</td><td>0.45 (0.20)</td><td>0.48 (0.17)</td><td>0.151</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Vmax (mM/s)</td><td>0.68 (0.17)</td><td>0.69 (0.24)</td><td>0.71 (0.17)</td><td>0.774</td><td>male<br>0.62 (0.10)</td><td>0.57 (0.11)</td><td>0.61 (0.09)</td><td>0.001</td><td>young<br>0.63 (0.11)</td><td>0.64 (0.17)</td><td>0.66 (0.14)</td><td>0.067</td><td>old<br>0.75 (0.20)</td><td>0.75 (0.30)</td><td>0.76 (0.18)</td><td>0.219</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>tPCrRec (s)</td><td>34.51 (7.65)</td><td>34.22 (8.93)</td><td>32.89 (7.15)</td><td>0.429</td><td>male<br>35.82 (8.24)</td><td>38.84 (7.21)</td><td>35.06 (6.71)</td><td>0.022</td><td>young<br>36.79 (7.33)</td><td>35.85 (9.42)</td><td>34.95 (7.89)</td><td>0.080</td><td>old<br>28.48 (5.33)</td><td>33.93 (9.91)</td><td>28.89 (6.63)</td><td>0.980</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>tPiRec (s)</td><td>27.60 (5.12)</td><td>31.13(8.53)**</td><td>26.85 (6.17)</td><td>0.0002</td><td>male<br>28.71 (4.63)</td><td>31.61 (7.65)</td><td>27.99 (5.85)</td><td>0.361</td><td>young<br>28.48 (5.33)</td><td>33.93 (9.91)</td><td>28.89 (6.63)</td><td>0.073</td><td>old<br>28.58 (4.87)</td><td>27.90 (5.30)</td><td>24.49 (4.80)</td><td>0.285</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>pH Rec</td><td>6.85 (0.03)</td><td>6.82 (0.03)**</td><td>6.85 (0.03)</td><td>&lt;0.0001</td><td>male<br>6.87 (0.02)</td><td>6.83 (0.03)</td><td>6.86 (0.03)</td><td>0.010</td><td>young<br>6.86 (0.03)</td><td>6.82 (0.03)</td><td>6.87 (0.02)</td><td>0.021</td><td>old<br>6.84 (0.03)</td><td>6.82 (0.03)</td><td>6.84 (0.03)*</td><td>0.271</td><td>0.501</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PCr repletion (%)</td><td>41.52 (9.96)</td><td>41.64 (8.32)</td><td>43.23 (8.75)</td><td>0.282</td><td>male<br>40.30 (10.37)</td><td>39.53 (9.30)</td><td>42.14 (9.11)</td><td>0.508</td><td>young<br>41.05 (10.19)</td><td>40.74 (8.73)</td><td>41.85 (8.85)</td><td>0.372</td><td>old<br>42.07 (10.09)</td><td>42.69 (8.04)</td><td>44.81 (8.71)</td><td>0.721</td><td>0.501</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>Table S7</caption><thead><tr><th></th><th>D-1</th><th>D+12</th><th>M+1</th></tr></thead><tbody><tr><td>T<sub>1PCr</sub> (s)</td><td>6.02 (0.49)</td><td>6.14 (0.49)</td><td>5.87 (0.68)</td></tr><tr><td>T<sub>1Pi</sub> (s)</td><td>5.48 (0.62)</td><td>5.27 (0.56)</td><td>5.26 (0.92)</td></tr><tr><td>T<sub>1γATP</sub> (s)</td><td>4.93 (0.43)</td><td>4.76 (0.53)</td><td>4.78 (0.62)</td></tr><tr><td>T<sub>1αATP</sub> (s)</td><td>3.3 (0.27)</td><td>3.18 (0.39)</td><td>3.23 (0.37)</td></tr><tr><td>T<sub>1βATP</sub> (s)</td><td>3.35 (0.36)</td><td>3.14 (0.54)</td><td>3.37 (0.57)</td></tr><tr><td>T<sub>1PDE</sub> (s)</td><td>8.95 (2.55)</td><td>8.77 (2.76)</td><td>8.54 (3.74)</td></tr><tr><td>[PCr]<sub>rest</sub> (mM)</td><td>33.91 (3.1)</td><td>33.21 (3.41)</td><td>32.92 (3.27)</td></tr><tr><td>[Pi]<sub>rest</sub> (mM)</td><td>4.04 (0.66)</td><td>3.87 (0.67)</td><td>4.13 (0.84)</td></tr><tr><td>[PDE]<sub>rest</sub> (mM)</td><td>3.04 (1.23)</td><td>2.82 (1.24)</td><td>3.08 (1.33)</td></tr><tr><td>[ADP]<sub>rest</sub> (μM)</td><td>6.66 (0.33)</td><td>6.37 (0.54)</td><td>6.65 (0.33)</td></tr><tr><td>[H<sub>2</sub>PO<sub>4</sub><sup>-</sup>]<sub>rest</sub> (mM)</td><td>1.71 (0.27)</td><td>1.7 (0.28)</td><td>1.76 (0.36)</td></tr><tr><td>pH<sub>rest</sub></td><td>6.88 (0.02)</td><td>6.86 (0.03)</td><td>6.88 (0.02)</td></tr><tr><td>PCr/Pi rest</td><td>8.55 (1.18)</td><td>8.78 (1.44)</td><td>8.21 (1.33)</td></tr><tr><td>Pi/PCr rest</td><td>0.12 (0.02)</td><td>0.12 (0.02)</td><td>0.12 (0.02)</td></tr><tr><td>T<sub>PCr</sub>Ex (s)*</td><td>30.48 (15.93)</td><td>32.94 (13.53)</td><td>28.71 (8.25)</td></tr><tr><td>T<sub>Pi</sub>Ex (s)*</td><td>24.2 (8.48)</td><td>25.19 (7.9)</td><td>23.1 (5.52)</td></tr><tr><td>[PCr]<sub>Post</sub> (mM)*</td><td>19.85 (3.89)</td><td>19.66 (3.39)</td><td>18.91 (3.55)</td></tr><tr><td>[Pi]<sub>Post</sub> (mM)*</td><td>13.93 (2.7)</td><td>14.22 (2.41)</td><td>15.08 (2.58)</td></tr><tr><td>[ATP]<sub>Post</sub> (mM)</td><td>8.1 (0.97)</td><td>8.32 (0.95)</td><td>8.31 (0.94)</td></tr><tr><td>[ADP]<sub>Post</sub> (μM)*</td><td>40.05 (16.42)</td><td>37.82 (12.58)</td><td>42.46 (14.66)</td></tr><tr><td>[H<sub>2</sub>PO<sub>4</sub><sup>-</sup>]<sub>Post</sub> (mM)*</td><td>5.88 (1.12)</td><td>6.15 (1.05)</td><td>6.35 (1.2)</td></tr><tr><td>pH<sub>Post</sub></td><td>6.89 (0.03)</td><td>6.87 (0.03)</td><td>6.89 (0.03)</td></tr><tr><td>pH change*</td><td>0.02 (0.02)</td><td>0.03 (0.02)</td><td>0.02 (0.02)</td></tr><tr><td>PCr Hydrolisis (%)</td><td>41.1 (11.33)</td><td>40.49 (9.27)</td><td>42.37 (10.27)</td></tr><tr><td>PCr change (mM)</td><td>13.93 (4.13)</td><td>13.47 (3.65)</td><td>14.02 (3.97)</td></tr><tr><td>Vi<sub>PCr</sub> (mM/s)*</td><td>0.41 (0.12)</td><td>0.41 (0.16)</td><td>0.44 (0.14)</td></tr><tr><td>V<sub>max</sub> (mM/s)*</td><td>0.68 (0.17)</td><td>0.69 (0.24)</td><td>0.71 (0.17)</td></tr><tr><td>T<sub>PCr</sub>Rec (s)</td><td>34.51 (7.85)</td><td>34.22 (8.93)</td><td>32.89 (7.15)</td></tr><tr><td>T<sub>Pi</sub>Rec (s)</td><td>27.6 (5.12)</td><td>31.13 (8.53)</td><td>26.85 (6.17)</td></tr><tr><td>[PCr]<sub>Rec</sub> (mM)</td><td>33.74 (3.2)</td><td>33.47 (4.36)</td><td>32.94 (3.32)</td></tr><tr><td>[Pi]<sub>Rec</sub> (mM)</td><td>2.93 (0.74)</td><td>2.95 (0.77)</td><td>2.99 (0.89)</td></tr><tr><td>[ATP]<sub>Rec</sub> (mM)*</td><td>8.42 (0.67)</td><td>8.53 (0.77)</td><td>8.35 (0.72)</td></tr><tr><td>[ADP]<sub>Rec</sub> (μM)*</td><td>6.65 (3.06)</td><td>6 (2.25)</td><td>6.19 (2.57)</td></tr><tr><td>[H<sub>2</sub>PO<sub>4</sub><sup>-</sup>]<sub>Rec</sub> (mM)</td><td>1.3 (0.34)</td><td>1.37 (0.37)</td><td>1.31 (0.39)</td></tr><tr><td>pH<sub>Rec</sub></td><td>6.85 (0.03)</td><td>6.82 (0.03)</td><td>6.85 (0.03)</td></tr><tr><td>PCr repletion (%)</td><td>41.52 (9.96)</td><td>41.64 (8.32)</td><td>43.23 (8.75)</td></tr><tr><td>PCr change (mM)*</td><td>13.98 (3.44)</td><td>14 (3.67)</td><td>14.21 (3.06)</td></tr></tbody></table>
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**Supplementary Material**

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