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Exercise Enhances Whole-Body Cholesterol Turnover in Mice


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Medicine & Science in Sports & Exercise: August 2010 - Volume 42 - Issue 8 - p 1460-1468
doi: 10.1249/MSS.0b013e3181cfcb02


Exercise is known as a preventative and therapeutic for cardiovascular diseases (10), but exactly howexercise exerts its beneficial actions on cardiovascular risk is largely unknown. One of the major hallmarks in exercise-induced risk reduction observed in human studies is an improvement in plasma lipid levels (6,11). Specifically, HDL levels increase with exercise, and concomitantly, total cholesterol, LDL cholesterol, and triglycerides decreased (6,11). Yet, the mechanisms behind such exercise-induced improvements in plasma lipids have not been defined. An enhanced transport of peripheral cholesterol toward the liver for subsequent secretion as free cholesterol or bile acids into bile and eventual excretion into feces has been suggested (18,27). In fact, this pathway, known as the reverse cholesterol transport (RCT), constitutes the major elimination route for excess cholesterol from the body (7). HDL is considered to be the carrier of cholesterol in the RCT pathway. To date, the effects of exercise on this pathway are poorly understood (18,27). However, if exercise enhances RCT, this should be reflected in an enhanced turnover of cholesterol and, possibly, modulations in bile acid metabolism.

Excess cholesterol can be removed from the body either as such or after conversion into bile acids. The liver secretes free cholesterol into bile, which is eventually released into the intestine, where it mixes with dietary cholesterol. Fractional absorption of cholesterol in humans shows a range between 30% and 70%, whereas in mice, values of 30%-50% are reported (5). Bile acids are synthesized from cholesterol exclusively in the liver and expelled into the intestinal lumen after ingestion of a meal. Bile acids are important molecules for the emulsification and reabsorption of fats in the intestine (22). The majority of bile acids are reabsorbed from the terminal ileum and transported back to the liver for resecretion into bile (enterohepatic circulation). The fraction of bile acids that escape reabsorption is lost in feces and constitutes an important part of cholesterol turnover because fecal bile acid loss is compensated for by de novo synthesis from cholesterol to maintain the bile acid pool size (9,19). This means that perturbations provoking an increased fecal bile acid loss also provoke an increased hepatic de novo bile acid synthesis.

Indeed, past limited work indicates that exercise potentially modulates cholesterol and bile acid metabolism (35,36,39). Early studies on female rats showed that 6 wk of voluntary wheel running promoted an increased biliary bile acid, cholesterol, and phospholipid secretion (39) and either increased or had no effect on bile flow (35,39). Moreover, a recent study in mice showed that 12 wk of forced treadmill exercise prevents gallstone formation in gallstone-prone mice (36). Gallstone disease is characterized by an abnormally high biliary cholesterol-to-bile acid and phospholipid ratio (22).

However, these limited data do not provide a clear understanding of the effects of exercise on bile acid and cholesterol turnover in rodents. Therefore, the purpose of this study was to clarify whether exercise modulates cholesterol and bile acid turnover in mice. If exercise indeed has such actions, this would offer novel insight in mechanisms contributing to the beneficial effects of exercise on cardiovascular diseases.


All experiments were approved by the Animal Care and Use Committee of the University of Groningen, The Netherlands. The University of Groningen is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (15), and experiments were performed under adherence to American College of Sports Medicine animal care standards.

Animals and Voluntary Cage Wheel Exercise

Ten-week-old male C57Bl6/J mice were purchased from Charles River Laboratories (L'Arbresle, France). On arrival at the animal facility, mice were housed singly in a cage (47 × 26 × 14.5 cm3) in a temperature-controlled room with a 12:12 light-dark cycle. Throughout the study, mice had access to standard commercial pelleted laboratory chow (RMH-B; Hope Farms BV, Woerden, The Netherlands) and water ad libitum. At 11 wk of age, mice were randomly selected to either voluntary cage wheel running (RUN) or to remain sedentary (SED). The voluntary running wheel setup used has been previously described (1). Briefly, the cage of RUN mice was equipped with a hamster-sized metal cage wheel with a diameter of 11 cm, which was fitted with a cycle computer containing a digital magnetic counter (Art No. K-13-TL SET-P3-NL; Xiron, Amsterdam, The Netherlands). Each morning, total distances run, total times run, and average and maximum daily speeds were recorded. Twice a week, mice were weighed, and food intake was recorded. Exposing mice to a voluntary running wheel for 2 wk has been previously shown to result in cardiac and skeletal muscle adaptations consistent with those of endurance exercise (1). Moreover, the voluntary cage wheel setup provides an antistress intervention (3) compared with forced exercise interventions that have been found to cause chronic stress-like changes in the hypothalamic-pituitary-adrenal axis (21).

Experimental Procedures

There were two primary experiments examining the effect of voluntary cage wheel running on cholesterol and bile acid metabolism: one on fecal, plasma, hepatic, and intestinal parameters and the other on biliary parameters. The end point of both experiments was reached after 2 wk of RUN or SED, at 13 wk of age.

Experiment 1: Determination of Plasma, Fecal, Hepatic, and Intestinal Parameters of Cholesterol and Bile Acid Metabolism

In experiment 1, RUN (n = 8) and SED (n = 6) mice were sacrificed by heart puncture under isoflurane anesthesia from 06:30 to 08:30 h. Plasma was stored at −20°C until analysis. The liver was quickly removed, weighed, and snap-frozen in liquid nitrogen. The small intestine was excised, flushed with ice-cold (4°C) PBS, divided into three sections of equal lengths, and, subsequently, snap-frozen in liquid nitrogen. Both liver and intestine were stored at −80°C for later biochemical analysis and RNA isolation.

Plasma and liver lipid analysis.

Plasma was collected by centrifugation of blood samples obtained via heart puncture. Plasma total cholesterol, free cholesterol, and triglyceride levels were measured by standard enzymatic methods using commercially available assay kits (Roche Diagnostics, Mannheim, Germany, and DiaSys Diagnostic Systems, Holzheim, Germany). Hepatic lipids were determined after extraction according to Bligh and Dyer (4) using the same commercially available kits as for plasma lipids.

We analyzed plasma plant sterol and lathosterol levels relative to plasma cholesterol levels as makers of intestinal cholesterol absorption (plant sterols) and cholesterol synthesis (lathosterol). The plasma plant sterol (campesterol + sitosterol)-cholesterol ratio is often used as a marker for intestinal cholesterol absorption because it correlates very well with rates of fractional intestinal cholesterol absorption (25). The plasma lathosterol-cholesterol ratio is a marker of cholesterol synthesis (25) because lathosterol is a precursor of the cholesterol biosynthetic pathway. Plasma plant sterol (campesterol and sitosterol) and lathosterol concentrations were determined by gas chromatography as described by Windler et al. (38). Pooled plasma samples from each group were used for lipoprotein separation by fast protein liquid chromatography on a Superose 6 column using an Akta Purifier (GE Healthcare, Diegem, Belgium).

Fecal parameters.

Forty-eight-hour feces productions were collected before running wheel exposure and at 2 wk of running wheel exposure. Feces were dried, weighed, and homogenized to a powder. Aliquots of fecal powder were used for analysis of total bile acids by an enzymatic fluorimetric assay (23). Neutral sterols and bile acid profiles were determined according to Arca et al. (2) and Setchell et al. (29), respectively.

RNA isolation and PCR procedures.

Total RNA was isolated from liver and intestine using TRI reagent (Sigma, St. Louis, MO) according to the manufacturer's protocol. cDNA was produced as described by Plosch et al. (28). Real-time PCR was performed on a 7900HT FAST real-time PCR system using FAST PCR Master Mix and MicroAmp FAST optical 96-well reaction plates (Applied Biosystems Europe, Nieuwerkerk aan den IJssel, The Netherlands). Primer and probe sequences have been published before ( PCR results were normalized to β-actin.

Experiment 2: Determination of Biliary Parameters of Cholesterol and Bile Acid Metabolism

Another set of eight RUN and five SED mice underwent gallbladder cannulation for collection of bile at 13 wk of age (28). Briefly, mice were anaesthetized by intraperitoneal injection with Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) (1 mL·kg−1) and diazepam (10 mg·kg−1). During the 30-min bile collection period, mice were placed in a humidified incubator to ensure maintenance of body temperature. Bile flow was determined gravimetrically, assuming a density of 1 g·mL−1 for bile. Bile was stored at −20°C until analysis. Total biliary bile acids were determined by an enzymatic fluorimetric assay (23). Levels of biliary cholesterol and phospholipids were measured as described by Kuipers et al. (17). Biliary bile acid composition was determined as described by Hulzebos et al. (14).


Statistical analysis was assessed using the Mann-Whitney U test (SPSS 12.0.1 for Windows, Chicago, IL). All data are expressed as means ± SD. A P value of <0.05 was accepted as statistically significant.


Basal parameters on 2 wk of voluntary wheel exercise.

Exposing 11-wk-old chow-fed C57Bl6/J mice to a voluntary running wheel for 2 wk did not affect body weight, liver weight, body weight-liver weight ratio, or intestinal length compared with sedentary mice (Table 1). Mice exposed to a running wheel averaged a daily distance of ∼10 km (Table 1) and were progressively running at a greater average speed and for a longer time and distance during the 2-wk running wheel exposure (see table, Supplemental Digital Content 1, which demonstrates an increase in running wheel performance during the 2-wk running intervention, Running mice consumed ∼30% more food than sedentary mice did (Table 1). Running mice had decreased total and free plasma cholesterol levels compared with sedentary controls (Table 2), but plasma lipoprotein profiles for cholesterol or triglycerides were not different between running and sedentary mice (see figure, Supplemental Digital Content 2, showing the plotted plasma lipoprotein profiles for the distribution of cholesterol (A) and triglycerides (B) in pooled plasma of sedentary and running mice, Hepatic total and free cholesterol stores were not affected by the running intervention (Table 2). It seems that there is a small, albeit not significant effect of running in decreasing hepatic esterified cholesterol content coinciding with a decrease in hepatic mRNA expression levels of Acat2 (see figure, Supplemental Digital Content 2, demonstrating a decrease in hepatic Acat2 mRNA levels on running,, the major cholesterol esterifying enzyme. In addition, running mice displayed approximately 20% lower hepatic triglyceride content compared with sedentary controls (Table 2).

Biometrical parameters and running wheel data.
Plasma and liver lipid levels.

Voluntary wheel exercise increased fecal neutral sterol and bile acid output.

To determine whether 2 wk of voluntary running wheel exercise alters cholesterol and bile acid metabolism in healthy chow-fed mice, we first assessed fecal parameters of cholesterol and bile acid metabolism. For this study, feces was collected during the last 48 h of the intervention in running and control mice to determine the fecal neutral sterol and bile acid output, as well as the fecal bile acid profile. We found that running mice had significantly increased fecal production and fecal neutral sterol and fecal bile acid output (all by ∼30%) compared with sedentary controls (Figs. 1A-C), whereas no differences in any of these parameters were observed between RUN and SED before the intervention (data not shown). Also, as shown in Table 3A, running mice had an increased fecal deoxycholate output compared with control mice. Deoxycholate is a secondary bile acid species that is formed from cholate in the intestine. The increase in fecal deoxycholate output in runners was paralleled by a nonsignificant decrease in fecal cholate output (P = 0.053). Thus, the total cholate output, which is the sum of cholate and deoxycholate, remained unaltered (Table 3A). Because under steady-state conditions fecal bile acid loss equals hepatic synthesis rate, these results imply that running wheel activity accelerates the conversion of cholesterol to bile acids in the liver.

Fecal parameters for SED (n = 6) and RUN (n = 8) after 2 wk of running: daily feces production (A), daily fecal bile acid output (B), and daily fecal neutral sterol output (C). *P < 0.05 versus SED, **P < 0.0.01 versus SED, ***P < 0.001 versus SED.
Fecal and biliary bile acid composition as percentage of total bile acids.

Voluntary running wheel exercise increases biliary bile acid secretion.

Observing this running-induced increase in fecal bile acid excretion, we next asked whether this increased loss was reflected in changes in biliary parameters. Thus, we subjected another set of mice of both groups to gallbladder cannulations for the collection of hepatic bile to examine the effect of voluntary running on bile flow, biliary bile acid, cholesterol, and phospholipid secretion. We found a trend for a higher bile flow in running mice (Fig. 2A). Then, as shown in Figure 2B, running mice had an increased biliary total bile acid secretion (Fig. 2B) and an increased absolute biliary bile acid concentration (27.78 ± 2.31 and 33.40 ± 4.52 mmol·L−1 for control and running mice, respectively, P = 0.042). However, secretion rates of biliary cholesterol and phospholipids were not affected by 2 wk of voluntary wheel running (Figs. 2C and D). Next, running increased the biliary total cholate-derived bile acid output because of an increase in biliary cholate output, although biliary deoxycholate remained unchanged (Table 3B). This increase in total cholates was reflected in an increased biliary cholate-chenodeoxycholate ratio upon running. Thus, the running-induced increase in fecal bile acid loss was paralleled by changes in biliary bile acid parameters.

Biliary parameters for SED (n = 5) and RUN (n = 8) after 2 wk of running: bile flow (A), biliary bile acid output (B), biliary cholesterol output (C), and biliary phospholipid output (D). *P < 0.05 versus SED.

We found that the running-induced fecal bile acid loss coincided with changes of some genes, but not others, implicated in bile acid transport in liver and intestine. For example, no changes were observed in ileal mRNA expression levels of Asbt, which functions in the active absorption of bile acids from the intestinal lumen into the ileal enterocytes (Fig. 3A), or Fgf15 (Fig. 3B), which is induced by bile acids in the ileum and represses bile acid synthesis via signaling mechanisms. Running mice displayed lower intestinal mRNA expressions of Ost alpha (Fig. 3C) and Ost beta (Fig. 3D), which act together in the basolateral efflux of bile acids from the ileum into the portal blood. Moreover, the hepatic mRNA expression levels of NTCP (Fig. 3E), a basolateral bile acid transporter acting to clear bile acids from the portal blood into the hepatocyte, were also decreased in running mice. On the other hand, mRNA expression levels of BSEP/Abcb11 (Fig. 3F), a transporter acting to transport bile acids from the hepatocyte into the bile, were not affected through the running intervention.

Quantitative RT-PCR of RNA from intestinal (A-D) and hepatic (E-F) bile acid transporters and signaling molecules in SED (n = 6) and RUN (n = 8) after 2 wk of running. A, Ileal apical sodium-dependent bile acid transporter, ASBT. B, Ileal fibroblast growth factor 15, Fgf15. Ileal organic solute transporter alpha (C) and beta (D). E, Hepatic sodium-dependent taurocholic cotransporting polypeptide, NTCP. F,Hepatic bile acid export pump, BSEP/Abcb11. *P < 0.05 versus SED.

Voluntary running wheel exercise decreases cholesterol absorption and increases cholesterol synthesis.

An increased neutral sterol loss is suggestive of a decreased reabsorption and an increased synthesis to compromise the consequences of the loss. We therefore questioned whether the increased fecal bile acid and neutral sterol loss was reflected by changes in markers of cholesterol reabsorption and synthesis. Indeed, the jejunal mRNA expression of Npc1l1, a protein required for intestinal cholesterol absorption (13), was decreased in running mice (Fig. 4A), whereas the jejunal mRNA expressions of the ATP-binding cassette transporters Abcg5 and Abcg8, two proteins implicated in control the of cholesterol absorption (8,40), were unchanged (Figs. 4B and C). Similar to the reduced Npc1l1 mRNA expression, the plasma total plant sterol-cholesterol ratio, a marker of cholesterol absorption, was decreased in running mice (Fig. 4E), thus indicating a decreased cholesterol absorption in runners. In addition, we also observed changes in markers of cholesterol synthesis indicative of an increased cholesterol synthesis on running. This is shown first in the running-induced increase in hepatic mRNA expression levels of HMGCoA reductase, a gene that encodes the rate-controlling enzyme in cholesterol synthesis (Fig. 5A), and second in the ∼60% increase in the plasma lathosterol-cholesterol ratio in running mice (Fig. 5B), supportive of an increased whole-body cholesterol synthesis upon running.

Parameters of cholesterol absorption in SED (n = 6) and RUN (n = 8) mice after 2 wk of running. Jejunal mRNA expression levels of: Niemann-Pick C1 Like 1, Npc1l1 (A); ATP-binding cassette transporter g5, Abcg5 (B); ATP-binding cassette transporter g8, Abcg8 (C); acyl CoA:cholesterol acyltransferase 2, Acat2 (D); and plasma total plant sterol-cholesterol ratio (E). *P < 0.05 versus SED.
Parameters of cholesterol metabolism in SED (n = 6) and RUN (n = 8) mice after 2 wk of running. A, Hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase, HMGCoA reductase, as a marker of cholesterol synthesis. B, Plasma lathosterol-cholesterol ratio as a marker of cholesterol. *P < 0.05 versus SED, **P < 0.001 versus SED.


This study was designed to assess whether voluntary wheel running modulates cholesterol and bile acid metabolism in healthy mice. The experiments presented here show that running modulates a variety of parameters of cholesterol and bile acid metabolism in mice and that these modulations result in an increased cholesterol turnover due to a decreased intestinal cholesterol and bile acid absorption. This has been demonstrated in three ways. First, the finding that running mice increased their fecal neutral sterol and bile acid output is consistent with a variety of parameters reflective of a decreased cholesterol absorption, the decreased plasma total plant sterol-cholesterol ratio (Fig. 4E), which correlates with the rate of cholesterol absorption (25), and the decreased jejunal expression of Npc1l1 (Fig. 4A), which is a protein required for intestinal cholesterol absorption. Second, the present data indicate that the running-induced fecal neutral sterol loss translated into an increased cholesterol synthesis (Figs. 5A and B), representing a physiological response to an enhanced cholesterol loss. Third, the increase in fecal bile acid output on running demonstrates an increased de novo bile acid synthesis, which contributes to cholesterol turnover.

Our observations of a running-induced increase in fecal production (31) and fecal neutral sterol and bile acid outputs (32) on one hand confirm some of the limited previous work on humans (30-32) but contradict others, showing that 4 months of training decreased the total fecal neutral sterol excretion in men (30) and that the total fecal bile acid secretion in male distance runners was lower due to an increased fecal production (31). The relevance of these findings is, however, difficult to evaluate because all previous studies were restricted to middle-aged male long-distance runners (30-32) and were not controlled for dietary intake, age (30-32), or endurance training (31,32), whereas the present investigation concerns a controlled study of previously sedentary but healthy mice having ad libitum but monitored access to standard laboratory chow and were exposed to a voluntary running wheel for 2 wk.

In our study, the loss of bile acids was specific for deoxycholate, a secondary bile acid (Table 3A). Deoxycholate is formed by the conversion of cholate, a primary bile acid, by intestinal flora (12). This increase in fecal deoxycholate is probably due to decreased bile acid reabsorption in the terminal ileum, thus allowing increased conversion by intestinal microbiota but also possibly by running-induced alterations in intestinal microbiota leading to an increased production of deoxycholate. Intriguingly, it has recently been shown that male rats exposed to a voluntary running wheel for 5 wk displayed a markedly changed composition of cecal microbiota (24). However, how exactly voluntary wheel running acts in provoking the specific increase in deoxycholate excretion in mice and what the role of intestinal microbiota is herein was not the scope of this study but deserves future investigation.

In addition to intestine-modulated changes in fecal bile acid composition, voluntary wheel running affects the intestine further by decreasing jejunal mRNA expression of Npc1l1, a protein critical for intestinal cholesterol absorption (13). Similarly, we found a running-induced decrease in jejunal acyl CoA:cholesterol acyltransferase 2 (Acat 2) mRNA expression levels (Fig. 4D). After cholesterol is taken up via Npc1l1 from the intestinal lumen to the enterocyte, it is esterified by Acat2 for proper chylomicron formation. The effect of voluntary running on decreasing intestinal cholesterol absorption was specific for the uptake transporter Npc1l1 because we show here that running did not affect mRNA levels of the cholesterol efflux transporters Abcg5 and Abcg8 in the intestine. Abcg5 and Abcg8 function as heterodimers and are responsible for sterol efflux from the enterocytes into the lumen, whereas Npc1l1 is involved in sterol entry from the lumen into the enterocyte (16). In support of a decreased cholesterol absorption despite no effects on Abcg5/8 expression, a previous study showed that reductions in cholesterol absorption correlated with decreased Npc1l1 mRNA expression, whereas Abcg5/8 mRNA expression remained unchanged (33).

Next, our study indicates in two ways that the exercise-induced malabsorption of sterols is compensated by an increased hepatic cholesterol synthesis. First, running mice had an increased hepatic mRNA expression level of HMGCoA reductase, the rate-limiting enzyme of cholesterol synthesis. And second, running mice had an increased plasma lathosterol-cholesterol ratio. Lathosterol is a precursor of cholesterol synthesis, and an increased lathosterol-cholesterol ratio is indicative of an increased cholesterol synthesis (25). However, our present results on young, healthy mice are in discrepancy with previous reports (34,37). The inconsistent and limited information that is available regarding the effects of exercise on cholesterol synthesis and absorption is largely restricted to plasma markers of cholesterol synthesis and absorption (34,37). These few previous investigations showed either only increases in plasma lathosterol-cholesterol (34) or only increases in plasma plant sterols-cholesterol (37) or no effect on markers of cholesterol synthesis or absorption at all (34). These discrepancies in markers of cholesterol absorption and synthesis between our study and previous works might be explained by differences in populations studied and exercise used. For example, previous works are limited to humans with lipoprotein abnormalities exercising three times a week for 8 wk or 6 months (34,37), whereas we used healthy mice that exercised every day for 2 wk.

Although our results indicate that voluntary wheel running increases bile acid synthesis, a surprising finding was that the expression of the main genes involved in bile acid synthesis, Cyp7a1 and Cyp27a1, was not affected by the intervention (see figure, Supplemental Digital Content 3, showing the hepatic mRNA expression levels of Cyp7a1 (A) and Cyp27a1 (B) in mice terminated between 06:30 and 08:00 h, Genes involved in bile acid synthesis follow a circadian rhythm (26). For this reason, we asked whether the time for termination and tissue collection originally chosen did not allow for an observation of differences in gene expression levels. However, we did not observe the anticipated running-induced increase in genes involved in bile acid synthesis when terminating another cohort of running mice and controls between 18:30 and 20:00 h (12 h later than all other mice described in this study; see figure, Supplemental Digital Content 3, showing the hepatic mRNA expression levels of Cyp7a1 (C) and Cyp27a1 (D)) in mice terminated between 18:30 and 20:00 h, Thus, within this study, the increase in bile acid synthesis on voluntary wheel running as demonstrated by fecal bile acid loss did not result in an up-regulation of mRNA expression levels of key genes involved in bile acid synthesis, specifically Cyp7a1. Possibly, a small decrease in reabsorbed intestinal bile acids is sufficient to repress the inhibitory signaling on Cyp7a1 without affecting mRNA expression levels in the voluntary wheel running intervention. In support of this, previous investigations on bile acid synthesis have shown that modulations of bile acid synthesis do not always correlate to changes in Cyp7a1 mRNA expression (14,20).

Unfortunately, there is a substantial lack of data describing the effects of exercise on parameters of cholesterol and bile acid metabolism coming from controlled human studies, which makes a translation of the results presented here into the human situation difficult. Most human studies are limited to fecal production rates and/or neutral sterol measurements in middle-aged trained men (30-32) or are limited to plasma lathosterol-cholesterol and plasma plant sterol-cholesterol levels in hypercholesterolemic patients (34,37). We thought that, for a more in-depth examination of the potential of exercise in modulating cholesterol and bile acid metabolism, a study setup that enabled the sampling of biliary, hepatic, and intestinal parameters, which are parameters difficult to obtain from healthy humans, is required.

In conclusion, the results presented here collectively show that voluntary wheel running in healthy chow-fed mice enhanced cholesterol turnover. The running-induced enhanced cholesterol turnover was reflected in a decreased intestinal cholesterol and bile acid absorption, leading to a subsequent increase in cholesterol synthesis and alterations in bile acid metabolism. Herein, this work offers novel findings on the mechanisms contributing to the beneficial effects of exercise on cardiovascular diseases.

The authors thank Vincent Bloks for critical reading of the manuscript.

The authors have no professional relationships to disclose with companies or manufacturers who will benefit from the results of the present study.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.


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©2010The American College of Sports Medicine