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Exercise-Induced Muscle Damage Is Not Attenuated by β-Hydroxy-β-Methylbutyrate and α-Ketoisocaproic Acid Supplementation

Nunan, David1; Howatson, Glyn2; van Someren, Ken A3

Journal of Strength and Conditioning Research: February 2010 - Volume 24 - Issue 2 - p 531-537
doi: 10.1519/JSC.0b013e3181c4d370
Original Research
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Nunan, D, Howatson, G, and van Someren, KA. Exercise-induced muscle damage is not attenuated by β-hydroxy-β-methylbutyrate and α-ketoisocaproic acid supplementation. J Strength Cond Res 24(2): 531-537, 2010-The purpose of this study was to examine the effects of combined oral β-hydroxy-β-methylbutyrate (HMB) and α-ketoisocaproic acid (KIC) supplementation on indices of exercise-induced muscle damage (EIMD) after an acute bout of eccentric-biased exercise. Fourteen male subjects were allocated to 2 groups: a placebo group (3 g·d−1 corn flour, N = 7) or an HMB + KIC group (3 g·d−1 HMB and 0.3 g·d−1 KIC, N = 7). Supplementation commenced 11 days before a 40-minute bout of downhill running and continued for 3 days post-exercise. Delayed-onset muscle soreness, mid-thigh girth, knee extensor range of motion, serum creatine kinase (CK) activity, and isometric and concentric torque were assessed pre-exercise and at 24, 48, and 72 hours post-exercise. Delayed-onset muscle soreness, CK activity, and isometric and concentric torque all changed over the 72-hour period (p < 0.05); however, HMB + KIC had no significant effect on any of the indices of muscle damage. Although 14 days HMB and KIC supplementation did not attenuate indices of EIMD after an acute bout of unaccustomed eccentric-biased exercise, there was a trend for a more rapid rate of recovery in isometric and isokinetic muscle function. β-hydroxy-β-methylbutyrate and KIC may therefore provide limited benefit in the recovery of muscle function after EIMD in untrained subjects or after unaccustomed exercise.

1School of Life Sciences, Kingston University, Kingston-upon-Thames, United Kingdom; 2School of Psychology and Sport Sciences, Northumbria University, Newcastle-upon-Tyne, United Kingdom; and 3English Institute of Sport, Bisham Abbey, United Kingdom

Address correspondence to Ken A. van Someren, ken.vansomeren@eis2win.co.uk.

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Introduction

β-hydroxy-β-methylbutyrate (HMB) is a downstream metabolite of the branch chain amino acid leucine via α-ketoisocaproate (KIC), the keto-analogue product of leucine (22). β-hydroxy-β-methylbutyrate is a widely recommended nutritional supplement for the attenuation of EIMD (2), and many commercially available HMB supplements also contain a small amount of KIC. Supplementation with dosages of 1.5 and 3.0 g·d−1 HMB has been shown to reduce muscle protein degradation and increase muscle mass after resistance exercise training for between 3 and 8 weeks (9,15,20), although some evidence suggests that these benefits are not observed in well-trained subjects (12,21).

Creatine kinase activity in blood, a commonly used marker of muscle damage (5,14), has also been shown to be reduced with HMB supplementation after resistance training (9,15,20) and prolonged running (11). Such protection against muscle damage has been attributed to the role of HMB in increasing cell membrane integrity through the provision of an additional source of cholesterol synthesis and therefore reducing damage-induced proteolysis (16); in addition, it has been hypothesized, although not yet tested, that HMB may act as a structural component within the cell (15).

Unaccustomed eccentric exercise has been widely reported to result in muscle damage with symptoms including reduced muscle function, increased pain and swelling, reduced range of motion (ROM), and elevated plasma creatine kinase (CK) activity (5,6,10,13). Recently, it has been shown that supplementation for 14 days with 3 g·d−1 HMB and 0.3 g·d−1 KIC reduces indices of muscle damage after a single bout of eccentric resistance exercise using the elbow flexors (23). Whole-body weight-bearing exercise is perhaps a better reflection of many sporting and recreational activities and therefore may provide a more ecologically valid exercise model with which to examine the effects of combined HMB and KIC supplementation. However, only one study (a published conference communication abstract) has investigated the effects of HMB supplementation on muscle damage after this type of exercise (3). This reported a marked reduction in muscle soreness 48 hours following 30 minutes downhill running after 28 days of 3 g·d−1 HMB and 3 g·d−1 HMB combined with 6 g·d−1 creatine supplementation. This study was limited by the assessment of only muscle soreness and isokinetic strength and the fact that subjects repeated downhill running trials before and after supplementation. Consequently, the well-established rapid adaptation of skeletal muscle to eccentric exercise and protection against subsequent eccentric exercise, known as the repeated bout effect (10,17,18), would have impacted on these findings.

Downhill running has been previously reported to elicit muscle damage (3,6), the signs and symptoms of which have been reported to be attenuated by HMB supplementation in other exercise models (11,20,23). We therefore hypothesized that 14 days HMB and KIC supplementation would reduce indices of muscle damage after an acute bout of downhill running.

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Methods

Experimental Approach to the Problem

The effects of HMB and KIC supplementation on EIMD after a bout of downhill running were compared with a placebo treatment in a single-blind experimental design. A between-subject design was adopted so as to avoid the influence of the repeated bout effect associated with a within-subject design (17). The supplement dose adopted and the dependent variables selected in this study were in accordance with previous research. A dose of 3 g·d−1 HMB with 0.3 g·d−1 KIC has been previously reported to significantly reduce the indices of muscle damage (23). The dependent variables have been investigated in a number of similar studies (3,19,23) and were measured before and at 24, 48 and 72 hours post-exercise and compared between treatments.

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Subjects

All experimental procedures were approved by the university's research ethics committee, and the study was conducted in accordance with the Declaration of Helsinki. Fourteen male volunteers participated in the study; all were recreational exercisers, but none was accustomed to eccentric-biased lower-limb resistance training or aerobic exercise (i.e., downhill running or bench stepping). Before participation, all subjects were informed about the investigation procedures and associated risks, completed a pre-test health-screening questionnaire, and provided written informed consent. Subjects had no recent history of having taken medication or nutritional supplements and were instructed to refrain from these for the duration of the study. Throughout the period of supplementation and testing, subjects were instructed to abstain from any form of unfamiliar and/or rigorous exercise that could potentially cause muscle damage. Adherence to these instructions was confirmed to the test investigator on each visit to the laboratory.

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β-hydroxy-β-methylbutyrate + α-ketoisocaproic Acid and Placebo Supplementation

Subjects were randomly assigned to either an HMB + KIC (N = 7; age = 30 ± 7 years, height = 178.0 ± 5.6 cm, body mass = 78.0 ± 7.0 kg [mean ± SD]) or placebo (N = 7; age = 30 ± 4 years, height = 183.3 ± 5.9 cm, body mass = 83.1 ± 10.7 kg [mean ± SD]) treatment in a single-blind design. The HMB + KIC group were prescribed 3 g·d−1 HMB with 0.3 g·d−1 KIC (Maximuscle HMB 1000; Maximuscle Ltd., Watford, UK); the placebo treatment comprised 3 g·d−1 corn flour. Each treatment was administered in the form of gelatin capsules taken 3 times daily in equal post-prandial doses. Supplementation was for 11 days before exercise and continued throughout the 72-hour period post-exercise, for a total of 14 days. All treatments were provided in individual sachets marked with the corresponding day of supplementation.

The purity of the supplement was analyzed by an independent laboratory using mass spectrometry-based analytical methods (Fourier transform near infrared spectroscopy using wavelengths scanning from 1000 to 2300 nm). A sample of the supplement was assessed for the presence of major classes of stimulants and anabolic steroids that have been implicated as contaminants in nutritional supplements (Table 1). These methods were accredited before sample analysis (ISO 17025), which is the required quality standard that underpins the World Anti-Doping Authority (WADA) testing procedures. The HMB + KIC capsules were homogenized and prepared for analyses using liquid-liquid and solid-phase extraction techniques. Stimulants were assessed by liquid chromatography, with mass spectrometric detection to a level of 500 ng·g−1 of supplement. Steroids were assessed by gas chromatography, with mass spectrometric detection to a level of 50 ng·g−1 of supplement.

Table 1

Table 1

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Downhill Running Protocol

On day 11 of the supplementation period, subjects performed a bout of intermittent downhill treadmill running designed to elicit muscle damage. The protocol was performed on a treadmill (Powerjog GXC200, Powersport International Ltd, United Kingdom) that was adapted to allow the belt to run in reverse. After a 5-minute, self-paced warm-up at 0% gradient, subjects performed 40 minutes downhill running at a speed of 11.2 km·h−1 (7 miles·h−1) and a gradient of 12%. The protocol consisted of 5 sets of 8-minute bouts of downhill running separated by 2 minutes of walking on a 0% gradient. This protocol was adapted from a previous study that used a similar protocol to elicit muscle damage (7).

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Dependent Variables

Measures of isometric and concentric torque, serum CK activity, delayed-onset muscle soreness (DOMS), ROM, and limb girth were taken pre-exercise and at 24, 48, and 72 hours post-exercise.

Isometric and concentric isokinetic torque: Isometric and isokinetic concentric peak torque assessment of the dominant leg extensors was conducted on a Biodex II isokinetic dynamometer (Biodex Medical Systems, Shirley, NY, USA). The mass of the exercising limb was measured for gravity correction; however, due to the difficulty in relaxing the muscle group in full extension, an angle of 30° knee flexion was used to avoid unwanted hamstring contribution (6).

Isometric torque was assessed at a knee angle of 80° flexion measured by goniometry. This knee angle has been shown to elicit maximal torque values for isometric knee extensor exercise (4). A period of familiarization and warm-up, consisting of 2 submaximal contractions and 1 maximal contraction, was given to all participants before the first measurement. After a 5-minute recovery period, participants were required to perform 3 maximum voluntary contractions of the quadriceps for approximately 3 seconds, with 10-second rest between repetitions. The highest peak torque from the 3 contractions was recorded. The coefficient of variation for isometric torque determined on consecutive days has been previously established as 1.0% in our laboratory.

Isokinetic concentric peak torque of the knee extensors was measured at angular velocities of 60 deg·s−1 and 180 deg·s−1. For both angular velocities, 3 maximal repetitions were performed from which the highest torque value was recorded. Standardized verbal encouragement was provided to the subject during all maximal contractions by the same test investigator; the dynamometer torque was displayed on the computer monitor in real time for feedback and motivational purposes (1). The coefficient of variation for peak torque at 60 deg·s−1 and 180 deg·s−1 determined on consecutive days has been previously established in our laboratory as 2.4 and 3.2%, respectively.

Serum CK activity: A venous blood sample was drawn from either the cephalic or the median cubital vein into a 5-ml serum separation tube; the serum was stored at −70°C for later analysis. Creatine kinase was determined using enzymatic dry slide chemistry (VITROS DT60II; Ortho-Clinical Diagnostics, Amersham, United Kingdom). Repeatability data from our laboratory for the intra-assay reliability show a coefficient of variation of <3%. Due to large intersubject variability in CK, values were logarithmic transformed (reported as LnCK). This reduced the heteroscedastic characteristics of the data and enabled the data to satisfy the homogeneity of variance assumption necessary for analysis of variance (ANOVA). Previous studies that have measured CK activity after damaging exercise have used a similar procedure (7,10).

Muscle soreness rating (DOMS): Knee extensor soreness was assessed using a 200-mm visual analogue scale ranging from “no soreness” to “extreme soreness” (10). The subject marked the scale according to the level of soreness when the knee was passively flexed and palpated. The marked level was measured with a standard ruler and the distance in millimeters from 0 mm to the mark was recorded.

Range of motion: Full flexion ROM was measured in the knee extensors of the dominant leg using a transparent plastic goniometer (Baseline; Physiomed, Cheshire, United Kingdom) when subjects lay supine. Total ROM was determined using a method previously described (8). All bony reference points were marked with permanent ink to ensure consistency of measurement on subsequent days. Intra-rater reliability for this method using technical error of measurement was 0.98%.

Limb girth: Limb girth of the dominant leg thigh was measured at the midpoint of the greater trochanter and the lateral epicondyle of the femur using a flexible anthropometric tape when the subject was standing fully relaxed in the anatomical position. The midpoint was marked with permanent ink to ensure consistent measures on subsequent days. Intra-rater reliability for this procedure was established at 0.27% using technical error of measurement.

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Statistical Analyses

Data are presented as mean ± SD values. Data for isometric and concentric torque, ROM, and limb girth are expressed as percentage change from pre-exercise levels; data for CK were log transformed before statistical analysis. All dependent variables were analyzed using a mixed factor (treatment, 2 × time, 4) repeated measures ANOVA and effect sizes (partial eta squared) were calculated for the treatment by time interaction effects. Mauchly's sphericity test was used to check the homogeneity of covariance; violations of the assumption of sphericity were corrected using the Greenhouse-Geisser adjustment. A significance level of p < 0.05 was established before analyses.

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Results

The results of the HMB + KIC analysis demonstrated 98% purity, the remaining 2% being primarily moisture. None of the stimulants or anabolic steroids screened for was found in the HMB + KIC supplement.

Figure 1 illustrates the percentage change in isometric (upper panel) and concentric torque at 60 deg·s−1 (middle panel) and at 180 deg·s−1 (lower panel) for the 2 treatment conditions. There was a significant decrease in isometric torque over the 72-hour period (F = 7.754, p < 0.01), decreasing by approximately 9% in both treatments at 24 hours post-exercise from baseline values of 248.3 ± 34.4 N·m and 290.2 ± 45.5 N·m in the HMB + KIC and placebo treatments, respectively. There was no significant difference between HMB + KIC and placebo treatments for isometric torque over the 72-hour period (effect size = 0.05); however, there was a trend for a more rapid recovery in the HMB + KIC treatment, with isometric torque being 0.8 ± 7.9% higher than baseline in HMB + KIC compared with 4.4 ± 5.8% lower than baseline in the placebo treatment at 72 hours post-exercise.

Figure 1

Figure 1

There was a significant decrease in both 60 deg·s−1 and 180 deg·s−1 concentric torque over the 72-hour period (F = 4.861, p < 0.01 and F = 5.366, p < 0.01 for 60 deg·s−1 and 180 deg·s−1 torque, respectively). Torque at 60 deg·s−1 decreased by 5.8 ± 12.8% and 7.5 ± 5.9% at 24 hours post-exercise from pre-exercise values of 202.8 ± 18.3 and 217.3 ± 23.7 N·m in HMB + KIC and placebo treatments, respectively. Torque at 180 deg·s−1 decreased by 5.8 ± 6.8% and 7.4 ± 4.2% at 24 hours post-exercise from pre-exercise values of 157.2 ± 14.2 and 160.8 ± 22.3 N·m in HMB + KIC and placebo treatments, respectively. There were no significant differences between treatments over time in the concentric torque measures (effect size = 0.04 and 0.06 for 60 deg·s−1 and 180 deg·s−1, respectively); however, there was a trend for a more rapid recovery in concentric torque at 60 deg·s−1 in the HMB + KIC treatment, with torque being 1.9 ± 7.2% higher than baseline in HMB + KIC compared with 2.5 ± 5.5% lower than baseline in the placebo treatment.

Results for CK and DOMS are shown in Figure 2 (upper and lower panels, respectively). Creatine kinase significantly increased after the exercise bout (F = 20.430, p < 0.001), reaching a peak at 24 hours post-exercise in both treatments (Figure 2, upper panel). Peak values for CK in the HMB + KIC and placebo treatments were 1479 ± 1398 and 1101 ± 1012 U·L−1 at 24 hours post-exercise, respectively, increasing from pre-exercise values of 251 ± 145 and 260 ± 158 U·L−1, respectively. There was no significant difference in CK between treatments over time (effect size = 0.03). There was an increase in DOMS after the exercise bout (F = 28.957, p < 0.001), which peaked at 48 hours post-exercise in both treatments (increasing to 55 ± 21 and 71 ± 47 mm in placebo and HMB + KIC treatments, respectively). There was no significant difference in DOMS between treatments over time (effect size = 0.10).

Figure 2

Figure 2

There was no change in ROM over the 72-hour period, and there were no differences between treatments over time (effect size = 0.07). The placebo treatment displayed a small, nonsignificant decrease in percentage ROM of 1% at 24 hours post-exercise, returning to pre-exercise levels at 72 hours (Figure 3, upper panel). The HMB + KIC treatment remained unchanged.

Figure 3

Figure 3

There was no significant change in limb girth over the 72-hour period and no significant difference between treatments over time (effect size = 0.23). Figure 3 (lower panel) illustrates a nonsignificant increase in limb girth for the placebo treatment at 24 and 48 hours post-exercise. The HMB + KIC treatment remained unchanged throughout the 72-hour period.

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Discussion

This is the first study to assess the effect of HMB and KIC supplementation on isometric and isokinetic torque, CK activity, muscle soreness, ROM, and limb girth after a bout of downhill running. Contrary to our hypothesis, supplementation for 14 days had no effect on any of the indices of muscle damage assessed in this study; however, there was a trend for a more rapid recovery of isometric function and isokinetic torque at 60 deg·s−1. Independent testing of the active ingredients in the HMB + KIC supplement demonstrated 98% purity, with the remaining 2% being mainly moisture and no traces of screened stimulants and steroids.

The exercise protocol used in this study was effective in eliciting muscle damage as shown by significant changes in CK, DOMS, and isometric and concentric torque after exercise. These results are similar to previous research identifying significant increases in CK and DOMS and reductions in torque after a bout of downhill running (7). There was, however, no change in ROM or limb girth after the exercise.

The effects of HMB and KIC supplementation reported in this study contrast with previous data from our laboratory (23), which found HMB and KIC supplementation for 14 days significantly reduces CK activity in blood, muscle soreness, and limb girth and attenuates the decrement in muscle function (1 repetition maximum) after a single bout of eccentric-biased resistance exercise. It is unclear why there is a discrepancy in results, given that we repeated the same supplementation strategy in the current study as that previously used (23); however, the current study adopted a downhill running protocol as opposed to eccentric repetitions of elbow flexion that were previously used (23). The nature of the exercise may account for the equivocal findings, although previous research has shown that downhill running comprises a significant eccentric component and elicits EIMD (6,7). The current results do however concur with others (19) who have examined the effects of HMB supplementation on muscle damage elicited by a single bout of damaging exercise. Paddon-Jones et al. (19) found HMB to have no effect on isometric, concentric, and eccentric muscle function; muscle soreness; or limb girth after an exercise protocol of 24 eccentric contractions; in contrast to our study, however, they supplemented with 40 mg·kg BM−1·d−1 for only 6 days before the exercise bout.

The only study to have examined the effects of HMB supplementation on muscle damage after downhill running is published solely in abstract form (3). The study only reported isokinetic muscle function and muscle soreness, showing HMB (3 g·d−1 for 28 days before exercise) and a combined HMB and creatine supplement (3 and 6 g·d−1, respectively, for 28 days before exercise) to significantly reduce muscle soreness at 48 hours after 30 minutes downhill running. HMB and creatine, both alone and in combination, also showed some evidence of attenuating the decrement in isokinetic muscle function. A major methodological limitation of the study by Byrd et al. (3) is that subjects performed a 30-minute bout of downhill running both before and after the 28-day supplementation period. It is well established that skeletal muscle demonstrates rapid adaptation to unaccustomed eccentric exercise, known as the repeated bout effect, whereby significantly less muscle damage is manifested when the same exercise is subsequently performed (10,17,18). This would almost certainly have contaminated the data of Byrd et al.'s (3) investigation. We have adopted a between-subject experimental design in this study to eliminate the repeated bout effect and therefore more accurately determine the effects of HMB and KIC supplementation.

Although no direct evidence exists as to exact mechanisms, HMB has been hypothesized to act as a precursor for cholesterol synthesis via its metabolism to β-hydroxy-β-methylglutaryl CoA, thus providing a carbon source for cholesterol synthesis (16). An increase in intracellular cholesterol may enhance cell membrane integrity and therefore reduce muscle damage after unaccustomed or intense exercise (16). Another hypothesis is that HMB serves as a structural component within the cell membrane (15). More recent research suggests that HMB may inhibit the ubiquitin-proteasome proteolytic pathway and stimulate protein synthesis; these proposed mechanisms of HMB action are not examined within this paper but have been recently reviewed elsewhere (24). The mechanisms by which KIC may reduce muscle damage are again unclear; however, given that KIC is a precursor to HMB, it is likely that it acts on the muscle cell in a similar manner. The results of the present study, however, do not confirm these hypotheses, as it is evident that HMB and KIC supplementation did not reduce muscle damage after a single bout of eccentric-biased downhill running.

In conclusion, and notwithstanding a nonsignificant trend for more rapid recovery of isometric function and isokinetic torque at 60 deg·s−1, 14 days supplementation with HMB and KIC did not reduce signs and symptoms associated with EIMD after a 30-minute bout of downhill running. Given the limited and equivocal nature of existing research in this area, further investigation is required to elucidate the effects and mechanisms of HMB and KIC supplementation on EIMD.

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Practical Applications

β-hydroxy-β-methylbutyrate is a popular nutritional supplement that is proposed to promote strength gains and reduce EIMD. Our findings indicate that HMB and KIC supplementation has no significant effect on indices of EIMD after an acute bout of unaccustomed eccentric-biased exercise; however, there was a trend for a more rapid rate of recovery in isometric and isokinetic muscle function. HMB and KIC may therefore provide some limited benefit in terms of recovery of muscle function after EIMD in untrained subjects or after unaccustomed exercise.

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Acknowledgments

This research was not funded by any external body and does not constitute endorsement of the product by the authors or the National Strength and Conditioning Association.

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Keywords:

eccentric exercise; downhill running; muscle soreness; muscle function

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