INFLUENCE OF MENSTRUAL CYCLE ON INDICES CONTRACTION-INDUCED MUSCLE DAMAGE MELISSA M. MARKOFSKI1
AND
OF
WILLIAM A. BRAUN2
1
Sealy Center on Aging, University of Texas Medical Branch, Galveston, Texas; and 2Department of Exercise Science, Shippensburg University, Shippensburg, Pennsylvania ABSTRACT
Markofski, MM and Braun, WA. Influence of menstrual cycle on indices of contraction-induced muscle damage. J Strength Cond Res 28(9): 2649–2656, 2014—Limited evidence suggests women exhibit a dampened response to contractioninduced muscle damage (CIMD). The purpose of this study was to examine if differences in symptoms of CIMD exist when induced in the menstrual cycle follicular or luteal phase. Sixteen resistance exercise trained women between the ages of 18–37 completed 75 eccentric-biased extension exercises with their nondominant arm. Creatine kinase (CK), elbow joint angles, arm volume, strength, and soreness were measured over 7 days. Estrogen was higher (p , 0.001) in the luteal group. The high estrogen group (luteal) had an overall greater strength decrement and higher CK concentration at 96 hours. Significant time effects were present for CK, elbow extension, elbow flexion, arm volume, and soreness. With the exception of strength and CK, signs and symptoms of CIMD were independent of menstrual cycle phase. Estrogen concentration in women may have limited effects on symptoms associated with muscle damage, but further research in this area is warranted.
KEY WORDS exercise, estrogens, creatine kinase, eccentric, woman
INTRODUCTION
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naccustomed exercise or exercise involving heavy eccentric bias is associated with contraction-induced muscle damage (CIMD). The soreness and damage associated with CIMD are temporary, but nevertheless unpleasant. Other transient side effects from muscle damage may include soreness, loss of range of motion, and reduction in strength (5). While CIMD can result from a variety of activities, in a laboratory setting eccentric contractions are frequently used to
Address correspondence to Melissa M. Markofski, memarkof@utmb. edu. 28(9)/2649–2656 Journal of Strength and Conditioning Research Ó 2014 National Strength and Conditioning Association
induce CIMD. However, even in a controlled laboratory situation, differences in participants’ response to CIMD can arise for a variety of reasons, including recent prior damage of the same muscle (11,22), activity level (21), type of activity (18), and health status (4). It has also been proposed that estrogen may exert a protective effect on muscle (14,30). Although some researchers have not found a sex-related difference in muscle damage (17,27), a number of recent studies (7,27,29,31,34) suggest a sex difference in response to eccentric loading, such that women may experience less muscle damage than men. If the sex difference is due to women having a higher concentration of circulating estrogen, then differences in response to eccentric muscle loading may exist within the normal hormonal fluctuations of a woman’s menstrual cycle. Muscle damage has many subjective measures, but an elevation of serum creatine kinase (CK) is often used as an objective indicator of muscle damage. Creatine kinase is present in muscle cells and an elevated presence in the serum after exercise indicates muscle damage (3,24). Muscle damage, irrespective of causation, will result in a loss of membrane integrity as reflected by CK release from cells. Creatine kinase concentration cannot quantify muscle damage but indicates its presence (12). Estrogen may confer protection against CIMD through 2 proposed mechanisms: by supporting integrity of cell membranes and by protecting the cell against free radical assault through antioxidant properties (9,10,19,20,30,35). For example, ovariectomized rats supplemented with estrogen had a lower plasma concentration of CK (30) and less calpain-like activity (34) post-CIMD than nonsupplemented rats. Additionally, an in vitro study of myoblasts found that the presence of estrogen promotes better maintenance of baseline ATP levels (34). This is important because better conservation of ATP homeostasis would offset the damageinduced increase of intracellular calcium ions, which precipitates the events leading to free radical assault on the cell. In addition to the free radical damage, calcium ions will also promote calpain-like activity (2,20,26). Ayres et al. (2) conducted several in vitro experiments to examine the protective role of estrogen against free radicals and other oxidants. The presence of estrogen significantly inhibited low-density lipoprotein oxidation and protected VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage against hydrogen peroxide–induced deoxyribonucleic acid damage. When estrogen was combined with other known free radical scavengers, protection was not magnified. Based on the results of these and other studies (1,6,26), it has been hypothesized that estrogen helps to stabilize cell membranes. Stupka et al. (31) investigated muscle damage differences between sexes in response to exercise performed at the same relative intensity. Statistical trends existed for sex differences in CK concentrations and leukocyte concentrations, which, when elevated, are indicative of an inflammatory response in damaged tissue. Both CK and leukocyte concentrations trended higher in men. Additionally, only the men had a significant elevation in circulating granulocyte concentrations, another measure of the inflammatory response. Because women have significantly higher circulating estrogen concentrations than men, the results of this study support the hypothesis that estrogen may confer a protective advantage against eccentric muscle work. These sex-related trends are unlikely to be related to differences in total muscle mass, which is often greater in men. Graves et al. (16) reported that although men generally have greater muscle mass than women, the volume of active muscle mass does not seem to influence the magnitude of change in CK in response to muscle damage. In the study of Graves et al. (16), a balanced-design was used to administer damage to 1 arm, both arms, or 1 leg. No significant differences in circulating CK were observed—thus concluding that the rise in CK is independent of the involved muscle area. Although studies using animals and men are important, they may not be representative of the response in human women. There is evidence to suggest that estrogen influences markers of CIMD; however, it is unknown if eccentric muscle actions, when performed during periods of high estrogen vs. low estrogen, will result in detectable differences in indices of muscle damage. Therefore, the purpose of this study was to examine if symptoms of muscle damage differ when eccentric loading is administered during the follicular vs. the luteal phase of the menstrual cycle. The luteal phase is associated with higher amounts of estrogen than the follicular phase. If estrogen confers a protective role, then muscle damage symptoms may differ between the 2 phases, with the luteal phase group experiencing less severe signs and symptoms of muscle damage.
METHODS Experimental Approach to the Problem
Regularly menstruating young women participated in an eccentric exercise bout on their nondominant arm. A repeated measure (phase 3 time) design was used to determine if there were menstrual cycle phase-dependent differences in response and recovery to CIMD. Soreness, arm volume, elbow extension, elbow flexion, and relaxed elbow joint angle were measured before the eccentric exercise and immediately, 24, 48, 72, 96, and 162 hours after exercise. Creatine kinase was measured before the eccentric exercise and 48, 72, 96, and 162 hours after exercise. Estradiol was measured to confirm menstrual cycle phase (Table 1).
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Subjects
Eighteen regularly menstruating resistance trained women between the ages of 18–38 years were recruited for this study. All subjects reported a regular menstrual cycle of between 21 and 35 days for at least the preceding 12 months. Although the cycle length was variable across subjects, within each subject the participant reported consistent cycle length, and this was used as a criterion for inclusion. Eight participants were hormonal birth control (HBC) pill users and the remaining 10 were not (non-HBC). Resistance exercise trained participants were employed in this study because they would have previous experience with muscle soreness and discomfort and would therefore better tolerate the extent of muscle damage. Each participant provided written informed consent, and all testing procedures and the informed consent were approved by the Institutional Review Board at California State Polytechnic University, Pomona. To be eligible to participate, volunteers had to engage in a minimum of 2 resistance training sessions (which included upper-body exercises) per week for a minimum of 3 continuous months preceding the study and were asked to refrain from resistance exercise while undergoing testing. Participants were not using nonsteroidal anti-inflammatory drugs (NSAIDs) and were free of upper-body injury in the past 6 months. All participants were tested within a 2-month window. To minimize variability, each participant reported to the laboratory at the same time of the day. The start dates of recent menstrual cycles and HBC use were self-reported. Phases of the menstrual cycle were confirmed by a verbal discussion with the participant. Participants randomized to the follicular group were tested on day 2 or 3 of the follicular phase, participants in the luteal phase were tested on day 1 or 2 of the luteal phase. Randomization into luteal or follicular phase groups was performed in a way that helped ensure equal distribution of HBC and non-HBC users. Participant characteristics are described in Table 1. Procedures
At least 4 days before the eccentric exercise bout, the participant reported to the laboratory for a 1 repetition maximum (1RM) biceps curl test on the nondominant arm. To help keep the joint angles the same for all trials, a standard preacher curl bench (Life Fitness, Inc., Schiller Park, IL, USA) was used for the 1RM testing, eccentric exercise bout, active soreness testing, and follow-up strength assessments. After a warm-up consisting of 8 repetitions using a light load, the maximum weight the participant could lift was determined. Weight was added to a dumbbell, in 2.5 lb (;1.14 kg) increments, until the participant could no longer lift the weight. Between each attempt, the participant was allowed 2 minutes of rest. The 1RM was used to determine the loads for eccentric exercise and for setting a test load to be employed during active soreness testing. On the day of the eccentric exercise session, baseline measurements were collected immediately before the
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a long water-filled freestanding plastic tube and displaced water TABLE 1. Subject descriptives (mean 6 SE). volume was measured. The measurement was repeated twice, Follicular phase Luteal phase Mean of subjects with a third trial if the measAge, y 25.9 6 2.2 23.7 6 2.5 24.9 6 1.6 urements varied by more than Height, cm 165.8 6 2.7 164.3 6 2.1 165.2 6 1.7 20 ml. A waterproof marker Weight, kg 65.4 6 5.2 67.7 6 3.2 66.3 6 3.1 was used to make a mark 1RM, kg 9.3 6 0.5 9.0 6 0.9 9.2 6 0.5 Estrogen, pg$ml21 77.3 6 10.7 187.8 6 27.8* 125.6 6 19.2 approximately over the inferior end of the deltoid muscle belly. *The luteal group had significantly (p = 0.0008) higher estrogen than the follicular group. The mark was reapplied daily to RM, repetition maximum. ensure that the participant was submerging her arm to the same depth every day. Relaxed elbow joint angle and passive flexion exercise. These included determination of baseline soreness, were measured with a goniometer. To measure relaxed elbow arm volume, a strength assessment (described below), and joint angle, the participant was instructed to relax her arm at relaxed elbow joint angle and passive flexion. In addition, a 5 ml her side; for elbow flexion, the participant held her upper arm venous blood sample was collected from an antecubital vein for parallel to the ground and let her forearm relax against her later determination of CK concentration and estrogen concenupper arm. tration (described below). The participants were instructed to The eccentric exercise bout consisted of 5 sets of 15 refrain from taking NSAIDs; performing any upper-body or repetitions each, with a 2-minute rest between each set. high-intensity lower-body weightlifting or downhill running; Because the eccentric exercise bout, the participant performed and icing, heating, stretching, or massaging the arm for the a light 2-set warm-up. The first set was 6–8 repetitions at ;30% duration of the study. of 1RM, followed by a second heavier warm-up (4–6 repetiThe active soreness rating was performed with a weight tions at ;60% 1RM). The first set of the eccentric muscle corresponding to approximately 40% of the participant’s action corresponded to 140% of the participant’s 1RM. The 1RM. After the participant raised and lowered the weight, participant was seated at the preacher curl bench to perform they indicated on a 10-cm visual analog scale how painful the exercise. An investigator assisted the participant in lifting the eccentric and concentric phases of the lift were for them the weight but did not aid in the eccentric work. A metroto perform. Arm volume was measured by water volumetry, nome was used to set the repetition rate of a 4-second eccenwhich is an established method for arm volume and swelling tric muscle action and 2-second aided concentric contraction. (32,36). Briefly, the participant would submerge her arm into Once the participant was no longer able to sustain a controlled 4-second eccentric muscle action, the weight was reduced to 130% of 1RM. Once this elicited failure, the load was further reduced to 120% of 1RM. All participants were able to complete all sets with a weight corresponding to at least 120% of their 1RM. The reduction of weight ensured that all participants were able to complete 75 contractions. All concentric strength measurements conducted after eccentric exercise were assessed as the percentage of the initial 1RM. The first attempt used a mass that was approximately 40% of the participant’s 1RM. After a 2-minute rest period, Figure 1. A moderate negative correlation was present between baseline estrogen concentration and strength recovery on day 7. Data points for 2 sets of subjects overlap closely, so they cannot be distinguished in this figure. the next attempt was at 50% of 1RM and continued to rise VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage
Figure 2. Strength recovery over the period of 1 week. A significant (p , 0.0001) main effect was present for time. *Strength was significantly higher than baseline at all other time points. †Follicular phase significantly (p = 0.009) higher strength than luteal phase. zFollicular phase significantly (p = 0.001) higher strength than luteal phase.
in increments of 10% until the participant was no longer able to lift the weight. Blood samples were collected from an antecubital vein from the nondamaged arm using sterile procedures. The samples were allowed to clot and subsequently centrifuged
at 3,000 rpm for 10 minutes. Serum was collected and stored at 2708 C for later analysis. Baseline estradiol was measured using an enzyme immunoassay kit (Diagnostic Systems Laboratories, Webster, TX, USA). Estradiol was measured to not only quantify estradiol,
Figure 3. A significant (p = 0.0008) time effect was present for relaxed elbow joint angle.
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but confirm phase. Following the instructions provided with the kit, 50 ml of the sample was analyzed and compared with standards analyzed at the same time as the sample. Estradiol was measured in duplicate. Creatine kinase was measured using a colorimetric analysis (Sigma Chemical Co., St Louis, MO, USA). In addition to the baseline sample, blood was collected 48, 72, 96, and 168 hours after the eccentric protocol. All other measurements were repeated immediately after the eccentric exercise bout and at 24, 48, 72, 96, and 168 hours postexercise. Statistical Analyses
*There was a time effect for arm volume, soreness, elbow flexion, and elbow extension.
Arm volume, ml 1642.6 6 115.3 1694.0 6 124.2 1702.0 6 130.1 1708.9 6 127.3 1750.3 6 118.8 1742.2 6 117.0 1666.9 6 129.7 Follicular 1701.3 6 91.0 1752.4 6 97.6 1730.9 6 100.7 1764.2 6 96.9 1781.8 6 101.8 1840.5 6 106.0 1778.1 6 94.2 Luteal Soreness, AU 1.77 6 0.8 19.4 6 4.6 54.6 6 8.7 56.9 6 7.5 39.3 6 8.5 23.4 6 7.9 6.1 6 2.9 Follicular 3.4 6 1.6 29.4 6 6.1 45.6 6 7.0 57.1 6 8.4 37.3 6 9.3 27.6 6 6.6 7.9 6 2.6 Luteal Elbow flexion, degrees 37.2 6 1.5 41.0 6 1.6 40.9 6 1.6 39.0 6 1.6 40.3 6 1.2 40.7 6 1.1 37.1 6 1.0 Follicular 42.6 6 4.6 43.7 6 3.1 43.9 6 3.6 41.8 6 2.1 40.7 6 2.9 38.9 6 2.7 42.7 6 3.8 Luteal Elbow extension, degrees 170.7 6 1.1 162.1 6 2.2 165.3 6 2.4 165.9 6 2.7 164.4 6 1.5 167.7 6 1.4 169.2 6 1.6 Follicular 171.1 6 1.1 166.1 6 2.7 162.9 6 2.8 163.6 6 1.6 163.3 6 2.3 166.0 6 2.2 169.6 6 1.6 Luteal
24 h Immediately post Baseline
TABLE 2. Arm volume, soreness, elbow flexion, and elbow extension (mean 6 SE).*
48 h
72 h
96 h
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All statistics were calculated with SAS v.9.1 (SAS Institute, Cary, North Carolina, USA). PROC GLM was used to determine possible baseline differences between groups. For repeated-measures analysis of variance (ANOVA), PROC MIXED was used. The data were evaluated for assumptions of normality (Shapiro-Wilkes), homogeneity of variance (residual plots), and independence of observations before evaluations of main effects and group interactions. As needed, a Box– Cox transformation was used to reduce skewness and stabilize variance. Hormonal birth control use was treated as a covariate. Creatine kinase measures were subjected to a 2 3 5 (phase 3 time) factor ANOVA. Arm volume, range of motion, relaxed elbow flexion, relaxed elbow joint angle, muscle soreness, and strength recovery were subjected to separate 2 3 7 (phase 3 time) factor ANOVA. Significant F ratios involving more than 2 means were followed by post hoc multiple pairwise comparisons using the Tukey method to locate the inequities. A criterion alpha level of 0.05 was adopted for all tests. Correlations were measured with Pearson’s product-moment correlation coefficient. All results are reported as mean and SEM (sx). Sample size was determined based on previous studies from this laboratory.
RESULTS Participants
During statistical analysis, 2 participants were removed from the data set due to incomplete data (1 noncompliance; 1 unrelated illness), the final sample size was 16 participants (follicular n = 9; luteal n = 7) and 6 of these participants were HBC users (follicular = 4; luteal = 2). Hormonal birth control use was determined to be a covariate and thus was treated as one in the statistical models. There were no statistically significant differences between follicular and luteal groups for any variable except estrogen. Estrogen Concentrations
The luteal group had significantly (p = 0.0008) higher estrogen concentration than the follicular group (187.8 6 27.8 vs. 77.3 6 10.7) (Table 1). A moderate negative correlation was present between baseline estrogen concentration and strength recovery, as calculated by percent of 1RM the participant was able to lift (Figure 1). VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage a significantly higher (p = 0.016) CK than the follicular group at 96 hours after (Figure 4). Creatine kinase remained elevated 168 hours post. No other effects or interactions were observed.
DISCUSSION Significant time effects for elbow flexion and extension, arm volume, soreness, and CK were present, indicating that the protocol elicited changes in common markers of muscle damage. However, a higher concentration of estrogen was not associated with a significant Figure 4. Creatine kinase concentration over time. A significant (p = 0.0014) time effect was present. *Luteal was improvement in signs and significantly (p = 0.016) higher than follicular phase at 96 hours. symptoms of CIMD. Contrary to the hypothesis, the participants’ physiological concenStrength Assessment trations of estrogen were not related to a protective effect Strength was evaluated as a percent change from the initial for the variables measured in this study. Based on the results 1RM. This allows for baseline strength to be taken into of the study, it seems likely that menstrual cycle phase has account without having to use baseline as a covariate. A little impact on CIMD in resistance training women. This significant (p , 0.0001) main effect was present for time. unexpected result may be partially explained by some recent Strength recovered over time, but full strength recovery did studies and some variations of studies in the present body of not occur during the 1-week course of the study (Figure 2). literature. A significant interaction (p = 0.04) was present, in that the Despite estrogen previously being shown to have a profollicular group had greater strength recovery at 96 hours after tective effect on cells (2,25,33,34), strength recovery was (p = 0.009) and at 168 hours after (p = 0.0013) than the luteal found to be better in the follicular (low estrogen) group. It group. was hypothesized that because estrogen appears to protect cells, then participants with higher estrogen concentrations Contraction-Induced Muscle Damage Exercise Bout would exhibit a less severe strength decrement; however, the The eccentric load and the number of eccentric repetitions opposite was observed in this study. A higher concentration performed at each percentage of 1RM (140%, 130%, 120%) of circulating estrogen was associated with poorer strength did not statistically differ between participant groups. recovery. The estrogen concentration at the time of damage Elbow Flexion and Extension may not have an effect on strength; instead, the estrogen A significant time effect was present for relaxed elbow joint concentrations during recovery may play a more crucial role. angle (Figure 3). A main effect for time (p = 0.04) was presThe days that the participants were tested were selected ent for elbow flexion (Table 2). No other effects or interacbecause of the anticipated difference of estrogen concentrations were observed. A main effect for time (p = 0.0002) was tions at those points in the menstrual cycle. During the present for elbow extension (Table 2). No other effects or recovery week, the estrogen concentrations of the follicular interactions were observed. group would be naturally increasing, whereas the luteal group would have revealed declining estrogen levels. Further Arm Volume and Soreness research of this question would be needed before a concluBecause of a large range of individual arm sizes, arm volume sion could be drawn. has been expressed as a percent change from baseline. A Postexercise elbow flexion and extension each showed main effect for time (p , 0.0001) was present. No other a significant time effect. There was also a significant time effects or interactions were observed for arm volume. A main effect for arm volume. These are common signs of muscle effect for time (p , 0.0001) was present for soreness. No damage (15) and were not unexpected. other effects or interactions were observed. Creatine kinase concentrations significantly increased Creatine Kinase across time, suggesting that the exercise caused enough A main effect for time (p = 0.0014) was present. There was CIMD to disrupt membrane integrity. It was surprising that also a significant interaction, with the luteal group showing the only interaction was the luteal group’s large CK
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Journal of Strength and Conditioning Research concentration at 96 hours later. We had hypothesized that the luteal group would have fewer signs of muscle damage, but found the opposite. The magnitude of difference at this time was also unexpected. One study that served as the driving force for the present study was conducted by Stupka et al. (31). Women in the follicular phase, who were taking HBC, were compared with men for signs and symptoms of CIMD. Following damage induction, the men had significantly higher circulating granulocyte concentrations than the women. Other trends were present that indicated that women may be less susceptible to muscle damage, but low power from a small sample size was identified as being a possible study limitation. A possible explanation for the lack of group differences in our study is that women, regardless of phase, may have sufficient estrogen present to offer some level of protection against muscle damage. Although this study does not directly provide evidence for this, a threshold for estrogen’s protective effect could exist. Ayres et al. (2) observed a potent antioxidant effect of estrogen, but they did not see any additive benefits when other known antioxidants were used in conjunction with estrogen. Along with the literature supporting a sex difference, the results of this study and the study of Ayres et al. indicate the effect of estrogen on signs and symptoms of muscle damage is a complex puzzle. It is possible that it is not just the amount of estrogen that has an effect on muscle damage, but also the interaction of estrogen with estrogen receptors. Other researchers (8,13,23) have reported sex differences in estrogen receptor activity, and the interaction of estrogen with estrogen receptors may be responsible for the sex difference response to CIMD that others have reported. Savage and Clarkson (28) compared muscle damage in women who did or did not use HBC. The only difference was that the birth control group had a delayed strength recovery. We treated birth control as a covariate and blocked for its use. However, 2 participants had to be excluded from the data set, and both of these participants were birth control users in the luteal group. This resulted in the follicular group having more HBC users. Despite this, the follicular group had greater strength recovery than the luteal group. Differences in response to CIMD vary between men and women, and the few published studies directly addressing this topic are in conflict. Participants in this study had a poorer strength recovery if they had higher estrogen at the time of the CIMD bout. This was in conflict with the hypothesis; yet, it is still possible that estrogen concentration influences CIMD outcomes. This area would benefit from additional research studies that further explore the complex role of estrogen in protection against CIMD, given the lengthy time course of events that are characteristic of muscle damage models.
PRACTICAL APPLICATIONS Based on the findings of this research, there is some indication that the response of women to a bout of CIMD
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is dependent on their menstrual cycle phase. This may have implications for women who do exhaustive or maximal weightlifting as part of their exercise training, in that their exercise program could be timed in accordance to their menstrual cycle. Specifically, that there may be some benefit to exhaustive or maximal weightlifting during the follicular, as opposed to the luteal, phase of the menstrual cycle. However, given the lack of strong evidence in support of a protective role of estrogen against muscle damage in the present study, it seems likely that menstrual phase will have small impact on symptoms of CIMD.
ACKNOWLEDGMENTS This work was conducted in the Department of Kinesiology and Health Promotion at California State Polytechnic University, Pomona, California No outside funding source was used to support this research. The results of the present study do not constitute endorsement of the product by the authors or the National Strength And Conditioning Association.
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24. Pen˜ailillo, L, Blazevich, A, Numazawa, H, and Nosaka, K. Metabolic and muscle damage profiles of concentric versus repeated eccentric cycling. Med Sci Sports Exerc 45: 1773–1781, 2013. 25. Persky, A, Green, P, Stubley, L, Howell, C, Zaulyanov, L, Brazeau, G, and Simpkins, J. Protective effect of estrogens against oxidative damage to heart and skeletal muscle in vivo and in vitro. Proc Soc Exp Biol Med 223: 59–66, 2000. 26. Razandi, M, Pedram, A, and Levin, E. Estrogen signals to the preservation of endothelial cell form and function. J Biol Chem 275: 38540–38546, 2000. 27. Rinard, J, Clarkson, P, Smith, L, and Grossman, M. Response of males and females to high-force eccentric exercise. J Sports Sci 18: 229–236, 2000. 28. Savage, K and Clarkson, P. Oral contraceptive use and exerciseinduced muscle damage and recovery. Contraception 66: 67–71, 2002. 29. Sayers, S and Clarkson, P. Force recovery after eccentric exercise in males and females. Eur J Appl Physiol 84: 122–126, 2001. 30. Sotiriadou, S, Kyparos, A, Mougios, V, Trontzos, C, Sidiras, G, and Matziari, C. Estrogen effect on some enzymes in female rats after downhill running. Physiol Res 52: 743–748, 2003. 31. Stupka, N, Lowther, S, Chorneyko, K, Bourgeois, J, Hogben, C, and Tarnopolsky, M. Gender differences in muscle inflammation after eccentric exercise. J Appl Physiol (1985) 89: 2325–2332, 2000. 32. Taylor, R, Jayasinghe, UW, Koelmeyer, L, Ung, O, and Boyages, J. Reliability and validity of arm volume measurements for assessment of lymphedema. Phys Ther 86: 205–214, 2006. 33. Tiidus, P, Holden, D, Bombardier, E, Zajchowski, S, Enns, D, and Belcastro, A. Estrogen effect on post-exercise skeletal muscle neutrophil infiltration and calpain activity. Can J Physiol Pharmacol 79: 400–406, 2001. 34. Tiidus, P. Influence of estrogen on skeletal muscle damage, inflammation, and repair. Exerc Sport Sci Rev 31: 40–44, 2003. 35. Wang, X, Sharma, R, Sikka, S, Thomas, AJ, Falcone, T, and Agarwal, A. Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 80: 531–535, 2003. 36. Waylett-Rendall, J and Seibly, D. A study of the accuracy of a commercially available volumeter. J Hand Ther 4: 10–13, 1991.
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AND
OF
WILLIAM A. BRAUN2
1
Sealy Center on Aging, University of Texas Medical Branch, Galveston, Texas; and 2Department of Exercise Science, Shippensburg University, Shippensburg, Pennsylvania ABSTRACT
Markofski, MM and Braun, WA. Influence of menstrual cycle on indices of contraction-induced muscle damage. J Strength Cond Res 28(9): 2649–2656, 2014—Limited evidence suggests women exhibit a dampened response to contractioninduced muscle damage (CIMD). The purpose of this study was to examine if differences in symptoms of CIMD exist when induced in the menstrual cycle follicular or luteal phase. Sixteen resistance exercise trained women between the ages of 18–37 completed 75 eccentric-biased extension exercises with their nondominant arm. Creatine kinase (CK), elbow joint angles, arm volume, strength, and soreness were measured over 7 days. Estrogen was higher (p , 0.001) in the luteal group. The high estrogen group (luteal) had an overall greater strength decrement and higher CK concentration at 96 hours. Significant time effects were present for CK, elbow extension, elbow flexion, arm volume, and soreness. With the exception of strength and CK, signs and symptoms of CIMD were independent of menstrual cycle phase. Estrogen concentration in women may have limited effects on symptoms associated with muscle damage, but further research in this area is warranted.
KEY WORDS exercise, estrogens, creatine kinase, eccentric, woman
INTRODUCTION
U
naccustomed exercise or exercise involving heavy eccentric bias is associated with contraction-induced muscle damage (CIMD). The soreness and damage associated with CIMD are temporary, but nevertheless unpleasant. Other transient side effects from muscle damage may include soreness, loss of range of motion, and reduction in strength (5). While CIMD can result from a variety of activities, in a laboratory setting eccentric contractions are frequently used to
Address correspondence to Melissa M. Markofski, memarkof@utmb. edu. 28(9)/2649–2656 Journal of Strength and Conditioning Research Ó 2014 National Strength and Conditioning Association
induce CIMD. However, even in a controlled laboratory situation, differences in participants’ response to CIMD can arise for a variety of reasons, including recent prior damage of the same muscle (11,22), activity level (21), type of activity (18), and health status (4). It has also been proposed that estrogen may exert a protective effect on muscle (14,30). Although some researchers have not found a sex-related difference in muscle damage (17,27), a number of recent studies (7,27,29,31,34) suggest a sex difference in response to eccentric loading, such that women may experience less muscle damage than men. If the sex difference is due to women having a higher concentration of circulating estrogen, then differences in response to eccentric muscle loading may exist within the normal hormonal fluctuations of a woman’s menstrual cycle. Muscle damage has many subjective measures, but an elevation of serum creatine kinase (CK) is often used as an objective indicator of muscle damage. Creatine kinase is present in muscle cells and an elevated presence in the serum after exercise indicates muscle damage (3,24). Muscle damage, irrespective of causation, will result in a loss of membrane integrity as reflected by CK release from cells. Creatine kinase concentration cannot quantify muscle damage but indicates its presence (12). Estrogen may confer protection against CIMD through 2 proposed mechanisms: by supporting integrity of cell membranes and by protecting the cell against free radical assault through antioxidant properties (9,10,19,20,30,35). For example, ovariectomized rats supplemented with estrogen had a lower plasma concentration of CK (30) and less calpain-like activity (34) post-CIMD than nonsupplemented rats. Additionally, an in vitro study of myoblasts found that the presence of estrogen promotes better maintenance of baseline ATP levels (34). This is important because better conservation of ATP homeostasis would offset the damageinduced increase of intracellular calcium ions, which precipitates the events leading to free radical assault on the cell. In addition to the free radical damage, calcium ions will also promote calpain-like activity (2,20,26). Ayres et al. (2) conducted several in vitro experiments to examine the protective role of estrogen against free radicals and other oxidants. The presence of estrogen significantly inhibited low-density lipoprotein oxidation and protected VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage against hydrogen peroxide–induced deoxyribonucleic acid damage. When estrogen was combined with other known free radical scavengers, protection was not magnified. Based on the results of these and other studies (1,6,26), it has been hypothesized that estrogen helps to stabilize cell membranes. Stupka et al. (31) investigated muscle damage differences between sexes in response to exercise performed at the same relative intensity. Statistical trends existed for sex differences in CK concentrations and leukocyte concentrations, which, when elevated, are indicative of an inflammatory response in damaged tissue. Both CK and leukocyte concentrations trended higher in men. Additionally, only the men had a significant elevation in circulating granulocyte concentrations, another measure of the inflammatory response. Because women have significantly higher circulating estrogen concentrations than men, the results of this study support the hypothesis that estrogen may confer a protective advantage against eccentric muscle work. These sex-related trends are unlikely to be related to differences in total muscle mass, which is often greater in men. Graves et al. (16) reported that although men generally have greater muscle mass than women, the volume of active muscle mass does not seem to influence the magnitude of change in CK in response to muscle damage. In the study of Graves et al. (16), a balanced-design was used to administer damage to 1 arm, both arms, or 1 leg. No significant differences in circulating CK were observed—thus concluding that the rise in CK is independent of the involved muscle area. Although studies using animals and men are important, they may not be representative of the response in human women. There is evidence to suggest that estrogen influences markers of CIMD; however, it is unknown if eccentric muscle actions, when performed during periods of high estrogen vs. low estrogen, will result in detectable differences in indices of muscle damage. Therefore, the purpose of this study was to examine if symptoms of muscle damage differ when eccentric loading is administered during the follicular vs. the luteal phase of the menstrual cycle. The luteal phase is associated with higher amounts of estrogen than the follicular phase. If estrogen confers a protective role, then muscle damage symptoms may differ between the 2 phases, with the luteal phase group experiencing less severe signs and symptoms of muscle damage.
METHODS Experimental Approach to the Problem
Regularly menstruating young women participated in an eccentric exercise bout on their nondominant arm. A repeated measure (phase 3 time) design was used to determine if there were menstrual cycle phase-dependent differences in response and recovery to CIMD. Soreness, arm volume, elbow extension, elbow flexion, and relaxed elbow joint angle were measured before the eccentric exercise and immediately, 24, 48, 72, 96, and 162 hours after exercise. Creatine kinase was measured before the eccentric exercise and 48, 72, 96, and 162 hours after exercise. Estradiol was measured to confirm menstrual cycle phase (Table 1).
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Subjects
Eighteen regularly menstruating resistance trained women between the ages of 18–38 years were recruited for this study. All subjects reported a regular menstrual cycle of between 21 and 35 days for at least the preceding 12 months. Although the cycle length was variable across subjects, within each subject the participant reported consistent cycle length, and this was used as a criterion for inclusion. Eight participants were hormonal birth control (HBC) pill users and the remaining 10 were not (non-HBC). Resistance exercise trained participants were employed in this study because they would have previous experience with muscle soreness and discomfort and would therefore better tolerate the extent of muscle damage. Each participant provided written informed consent, and all testing procedures and the informed consent were approved by the Institutional Review Board at California State Polytechnic University, Pomona. To be eligible to participate, volunteers had to engage in a minimum of 2 resistance training sessions (which included upper-body exercises) per week for a minimum of 3 continuous months preceding the study and were asked to refrain from resistance exercise while undergoing testing. Participants were not using nonsteroidal anti-inflammatory drugs (NSAIDs) and were free of upper-body injury in the past 6 months. All participants were tested within a 2-month window. To minimize variability, each participant reported to the laboratory at the same time of the day. The start dates of recent menstrual cycles and HBC use were self-reported. Phases of the menstrual cycle were confirmed by a verbal discussion with the participant. Participants randomized to the follicular group were tested on day 2 or 3 of the follicular phase, participants in the luteal phase were tested on day 1 or 2 of the luteal phase. Randomization into luteal or follicular phase groups was performed in a way that helped ensure equal distribution of HBC and non-HBC users. Participant characteristics are described in Table 1. Procedures
At least 4 days before the eccentric exercise bout, the participant reported to the laboratory for a 1 repetition maximum (1RM) biceps curl test on the nondominant arm. To help keep the joint angles the same for all trials, a standard preacher curl bench (Life Fitness, Inc., Schiller Park, IL, USA) was used for the 1RM testing, eccentric exercise bout, active soreness testing, and follow-up strength assessments. After a warm-up consisting of 8 repetitions using a light load, the maximum weight the participant could lift was determined. Weight was added to a dumbbell, in 2.5 lb (;1.14 kg) increments, until the participant could no longer lift the weight. Between each attempt, the participant was allowed 2 minutes of rest. The 1RM was used to determine the loads for eccentric exercise and for setting a test load to be employed during active soreness testing. On the day of the eccentric exercise session, baseline measurements were collected immediately before the
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a long water-filled freestanding plastic tube and displaced water TABLE 1. Subject descriptives (mean 6 SE). volume was measured. The measurement was repeated twice, Follicular phase Luteal phase Mean of subjects with a third trial if the measAge, y 25.9 6 2.2 23.7 6 2.5 24.9 6 1.6 urements varied by more than Height, cm 165.8 6 2.7 164.3 6 2.1 165.2 6 1.7 20 ml. A waterproof marker Weight, kg 65.4 6 5.2 67.7 6 3.2 66.3 6 3.1 was used to make a mark 1RM, kg 9.3 6 0.5 9.0 6 0.9 9.2 6 0.5 Estrogen, pg$ml21 77.3 6 10.7 187.8 6 27.8* 125.6 6 19.2 approximately over the inferior end of the deltoid muscle belly. *The luteal group had significantly (p = 0.0008) higher estrogen than the follicular group. The mark was reapplied daily to RM, repetition maximum. ensure that the participant was submerging her arm to the same depth every day. Relaxed elbow joint angle and passive flexion exercise. These included determination of baseline soreness, were measured with a goniometer. To measure relaxed elbow arm volume, a strength assessment (described below), and joint angle, the participant was instructed to relax her arm at relaxed elbow joint angle and passive flexion. In addition, a 5 ml her side; for elbow flexion, the participant held her upper arm venous blood sample was collected from an antecubital vein for parallel to the ground and let her forearm relax against her later determination of CK concentration and estrogen concenupper arm. tration (described below). The participants were instructed to The eccentric exercise bout consisted of 5 sets of 15 refrain from taking NSAIDs; performing any upper-body or repetitions each, with a 2-minute rest between each set. high-intensity lower-body weightlifting or downhill running; Because the eccentric exercise bout, the participant performed and icing, heating, stretching, or massaging the arm for the a light 2-set warm-up. The first set was 6–8 repetitions at ;30% duration of the study. of 1RM, followed by a second heavier warm-up (4–6 repetiThe active soreness rating was performed with a weight tions at ;60% 1RM). The first set of the eccentric muscle corresponding to approximately 40% of the participant’s action corresponded to 140% of the participant’s 1RM. The 1RM. After the participant raised and lowered the weight, participant was seated at the preacher curl bench to perform they indicated on a 10-cm visual analog scale how painful the exercise. An investigator assisted the participant in lifting the eccentric and concentric phases of the lift were for them the weight but did not aid in the eccentric work. A metroto perform. Arm volume was measured by water volumetry, nome was used to set the repetition rate of a 4-second eccenwhich is an established method for arm volume and swelling tric muscle action and 2-second aided concentric contraction. (32,36). Briefly, the participant would submerge her arm into Once the participant was no longer able to sustain a controlled 4-second eccentric muscle action, the weight was reduced to 130% of 1RM. Once this elicited failure, the load was further reduced to 120% of 1RM. All participants were able to complete all sets with a weight corresponding to at least 120% of their 1RM. The reduction of weight ensured that all participants were able to complete 75 contractions. All concentric strength measurements conducted after eccentric exercise were assessed as the percentage of the initial 1RM. The first attempt used a mass that was approximately 40% of the participant’s 1RM. After a 2-minute rest period, Figure 1. A moderate negative correlation was present between baseline estrogen concentration and strength recovery on day 7. Data points for 2 sets of subjects overlap closely, so they cannot be distinguished in this figure. the next attempt was at 50% of 1RM and continued to rise VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage
Figure 2. Strength recovery over the period of 1 week. A significant (p , 0.0001) main effect was present for time. *Strength was significantly higher than baseline at all other time points. †Follicular phase significantly (p = 0.009) higher strength than luteal phase. zFollicular phase significantly (p = 0.001) higher strength than luteal phase.
in increments of 10% until the participant was no longer able to lift the weight. Blood samples were collected from an antecubital vein from the nondamaged arm using sterile procedures. The samples were allowed to clot and subsequently centrifuged
at 3,000 rpm for 10 minutes. Serum was collected and stored at 2708 C for later analysis. Baseline estradiol was measured using an enzyme immunoassay kit (Diagnostic Systems Laboratories, Webster, TX, USA). Estradiol was measured to not only quantify estradiol,
Figure 3. A significant (p = 0.0008) time effect was present for relaxed elbow joint angle.
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but confirm phase. Following the instructions provided with the kit, 50 ml of the sample was analyzed and compared with standards analyzed at the same time as the sample. Estradiol was measured in duplicate. Creatine kinase was measured using a colorimetric analysis (Sigma Chemical Co., St Louis, MO, USA). In addition to the baseline sample, blood was collected 48, 72, 96, and 168 hours after the eccentric protocol. All other measurements were repeated immediately after the eccentric exercise bout and at 24, 48, 72, 96, and 168 hours postexercise. Statistical Analyses
*There was a time effect for arm volume, soreness, elbow flexion, and elbow extension.
Arm volume, ml 1642.6 6 115.3 1694.0 6 124.2 1702.0 6 130.1 1708.9 6 127.3 1750.3 6 118.8 1742.2 6 117.0 1666.9 6 129.7 Follicular 1701.3 6 91.0 1752.4 6 97.6 1730.9 6 100.7 1764.2 6 96.9 1781.8 6 101.8 1840.5 6 106.0 1778.1 6 94.2 Luteal Soreness, AU 1.77 6 0.8 19.4 6 4.6 54.6 6 8.7 56.9 6 7.5 39.3 6 8.5 23.4 6 7.9 6.1 6 2.9 Follicular 3.4 6 1.6 29.4 6 6.1 45.6 6 7.0 57.1 6 8.4 37.3 6 9.3 27.6 6 6.6 7.9 6 2.6 Luteal Elbow flexion, degrees 37.2 6 1.5 41.0 6 1.6 40.9 6 1.6 39.0 6 1.6 40.3 6 1.2 40.7 6 1.1 37.1 6 1.0 Follicular 42.6 6 4.6 43.7 6 3.1 43.9 6 3.6 41.8 6 2.1 40.7 6 2.9 38.9 6 2.7 42.7 6 3.8 Luteal Elbow extension, degrees 170.7 6 1.1 162.1 6 2.2 165.3 6 2.4 165.9 6 2.7 164.4 6 1.5 167.7 6 1.4 169.2 6 1.6 Follicular 171.1 6 1.1 166.1 6 2.7 162.9 6 2.8 163.6 6 1.6 163.3 6 2.3 166.0 6 2.2 169.6 6 1.6 Luteal
24 h Immediately post Baseline
TABLE 2. Arm volume, soreness, elbow flexion, and elbow extension (mean 6 SE).*
48 h
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96 h
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All statistics were calculated with SAS v.9.1 (SAS Institute, Cary, North Carolina, USA). PROC GLM was used to determine possible baseline differences between groups. For repeated-measures analysis of variance (ANOVA), PROC MIXED was used. The data were evaluated for assumptions of normality (Shapiro-Wilkes), homogeneity of variance (residual plots), and independence of observations before evaluations of main effects and group interactions. As needed, a Box– Cox transformation was used to reduce skewness and stabilize variance. Hormonal birth control use was treated as a covariate. Creatine kinase measures were subjected to a 2 3 5 (phase 3 time) factor ANOVA. Arm volume, range of motion, relaxed elbow flexion, relaxed elbow joint angle, muscle soreness, and strength recovery were subjected to separate 2 3 7 (phase 3 time) factor ANOVA. Significant F ratios involving more than 2 means were followed by post hoc multiple pairwise comparisons using the Tukey method to locate the inequities. A criterion alpha level of 0.05 was adopted for all tests. Correlations were measured with Pearson’s product-moment correlation coefficient. All results are reported as mean and SEM (sx). Sample size was determined based on previous studies from this laboratory.
RESULTS Participants
During statistical analysis, 2 participants were removed from the data set due to incomplete data (1 noncompliance; 1 unrelated illness), the final sample size was 16 participants (follicular n = 9; luteal n = 7) and 6 of these participants were HBC users (follicular = 4; luteal = 2). Hormonal birth control use was determined to be a covariate and thus was treated as one in the statistical models. There were no statistically significant differences between follicular and luteal groups for any variable except estrogen. Estrogen Concentrations
The luteal group had significantly (p = 0.0008) higher estrogen concentration than the follicular group (187.8 6 27.8 vs. 77.3 6 10.7) (Table 1). A moderate negative correlation was present between baseline estrogen concentration and strength recovery, as calculated by percent of 1RM the participant was able to lift (Figure 1). VOLUME 28 | NUMBER 9 | SEPTEMBER 2014 |
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Menstrual Cycle and Muscle Damage a significantly higher (p = 0.016) CK than the follicular group at 96 hours after (Figure 4). Creatine kinase remained elevated 168 hours post. No other effects or interactions were observed.
DISCUSSION Significant time effects for elbow flexion and extension, arm volume, soreness, and CK were present, indicating that the protocol elicited changes in common markers of muscle damage. However, a higher concentration of estrogen was not associated with a significant Figure 4. Creatine kinase concentration over time. A significant (p = 0.0014) time effect was present. *Luteal was improvement in signs and significantly (p = 0.016) higher than follicular phase at 96 hours. symptoms of CIMD. Contrary to the hypothesis, the participants’ physiological concenStrength Assessment trations of estrogen were not related to a protective effect Strength was evaluated as a percent change from the initial for the variables measured in this study. Based on the results 1RM. This allows for baseline strength to be taken into of the study, it seems likely that menstrual cycle phase has account without having to use baseline as a covariate. A little impact on CIMD in resistance training women. This significant (p , 0.0001) main effect was present for time. unexpected result may be partially explained by some recent Strength recovered over time, but full strength recovery did studies and some variations of studies in the present body of not occur during the 1-week course of the study (Figure 2). literature. A significant interaction (p = 0.04) was present, in that the Despite estrogen previously being shown to have a profollicular group had greater strength recovery at 96 hours after tective effect on cells (2,25,33,34), strength recovery was (p = 0.009) and at 168 hours after (p = 0.0013) than the luteal found to be better in the follicular (low estrogen) group. It group. was hypothesized that because estrogen appears to protect cells, then participants with higher estrogen concentrations Contraction-Induced Muscle Damage Exercise Bout would exhibit a less severe strength decrement; however, the The eccentric load and the number of eccentric repetitions opposite was observed in this study. A higher concentration performed at each percentage of 1RM (140%, 130%, 120%) of circulating estrogen was associated with poorer strength did not statistically differ between participant groups. recovery. The estrogen concentration at the time of damage Elbow Flexion and Extension may not have an effect on strength; instead, the estrogen A significant time effect was present for relaxed elbow joint concentrations during recovery may play a more crucial role. angle (Figure 3). A main effect for time (p = 0.04) was presThe days that the participants were tested were selected ent for elbow flexion (Table 2). No other effects or interacbecause of the anticipated difference of estrogen concentrations were observed. A main effect for time (p = 0.0002) was tions at those points in the menstrual cycle. During the present for elbow extension (Table 2). No other effects or recovery week, the estrogen concentrations of the follicular interactions were observed. group would be naturally increasing, whereas the luteal group would have revealed declining estrogen levels. Further Arm Volume and Soreness research of this question would be needed before a concluBecause of a large range of individual arm sizes, arm volume sion could be drawn. has been expressed as a percent change from baseline. A Postexercise elbow flexion and extension each showed main effect for time (p , 0.0001) was present. No other a significant time effect. There was also a significant time effects or interactions were observed for arm volume. A main effect for arm volume. These are common signs of muscle effect for time (p , 0.0001) was present for soreness. No damage (15) and were not unexpected. other effects or interactions were observed. Creatine kinase concentrations significantly increased Creatine Kinase across time, suggesting that the exercise caused enough A main effect for time (p = 0.0014) was present. There was CIMD to disrupt membrane integrity. It was surprising that also a significant interaction, with the luteal group showing the only interaction was the luteal group’s large CK
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Journal of Strength and Conditioning Research concentration at 96 hours later. We had hypothesized that the luteal group would have fewer signs of muscle damage, but found the opposite. The magnitude of difference at this time was also unexpected. One study that served as the driving force for the present study was conducted by Stupka et al. (31). Women in the follicular phase, who were taking HBC, were compared with men for signs and symptoms of CIMD. Following damage induction, the men had significantly higher circulating granulocyte concentrations than the women. Other trends were present that indicated that women may be less susceptible to muscle damage, but low power from a small sample size was identified as being a possible study limitation. A possible explanation for the lack of group differences in our study is that women, regardless of phase, may have sufficient estrogen present to offer some level of protection against muscle damage. Although this study does not directly provide evidence for this, a threshold for estrogen’s protective effect could exist. Ayres et al. (2) observed a potent antioxidant effect of estrogen, but they did not see any additive benefits when other known antioxidants were used in conjunction with estrogen. Along with the literature supporting a sex difference, the results of this study and the study of Ayres et al. indicate the effect of estrogen on signs and symptoms of muscle damage is a complex puzzle. It is possible that it is not just the amount of estrogen that has an effect on muscle damage, but also the interaction of estrogen with estrogen receptors. Other researchers (8,13,23) have reported sex differences in estrogen receptor activity, and the interaction of estrogen with estrogen receptors may be responsible for the sex difference response to CIMD that others have reported. Savage and Clarkson (28) compared muscle damage in women who did or did not use HBC. The only difference was that the birth control group had a delayed strength recovery. We treated birth control as a covariate and blocked for its use. However, 2 participants had to be excluded from the data set, and both of these participants were birth control users in the luteal group. This resulted in the follicular group having more HBC users. Despite this, the follicular group had greater strength recovery than the luteal group. Differences in response to CIMD vary between men and women, and the few published studies directly addressing this topic are in conflict. Participants in this study had a poorer strength recovery if they had higher estrogen at the time of the CIMD bout. This was in conflict with the hypothesis; yet, it is still possible that estrogen concentration influences CIMD outcomes. This area would benefit from additional research studies that further explore the complex role of estrogen in protection against CIMD, given the lengthy time course of events that are characteristic of muscle damage models.
PRACTICAL APPLICATIONS Based on the findings of this research, there is some indication that the response of women to a bout of CIMD
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is dependent on their menstrual cycle phase. This may have implications for women who do exhaustive or maximal weightlifting as part of their exercise training, in that their exercise program could be timed in accordance to their menstrual cycle. Specifically, that there may be some benefit to exhaustive or maximal weightlifting during the follicular, as opposed to the luteal, phase of the menstrual cycle. However, given the lack of strong evidence in support of a protective role of estrogen against muscle damage in the present study, it seems likely that menstrual phase will have small impact on symptoms of CIMD.
ACKNOWLEDGMENTS This work was conducted in the Department of Kinesiology and Health Promotion at California State Polytechnic University, Pomona, California No outside funding source was used to support this research. The results of the present study do not constitute endorsement of the product by the authors or the National Strength And Conditioning Association.
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