Two reliable circulating proteins useful for skeletal muscle damage evaluation are creatine kinase (CK) and myoglobin (Mb). Serum CK increases more slowly than Mb concentrations post-exercise (12), and Mb is considered a more sensitive marker of skeletal muscle damage (12). Normally an acute bout of intense resistance exercise increases serum CK and Mb concentrations, but their responses can be modulated by training status (5,9,22), exercise mode (7,34), and fluid ingestion (6,32).
Athletes intensely focus their training to improve muscle mass and performance. Often these athletes fail to properly rehydrate post-exercise (15,21,24). We do not know how chronic hypohydration may affect the different mechanical and metabolic stresses of resistance exercise and in turn affect the extent of imposed muscle damage.
The effect of hydration state on muscle damage has not been studied thoroughly. Greiwe et al. (16) found that dehydration (to −4%) in combination with heat stress had no significant effect on Mb concentrations, but exercise per se was not examined. When exercise was initiated in a euhydrated state, fluid ingestion attenuated the release of CK and Mb post-exercise (6,32). Seifert et al. (32) reported significantly increased CK and Mb concentrations after Alpine skiing without fluid ingestion versus noncaloric fluid ingestion, which suggested dehydration may have produced a metabolic stress great enough to induce muscle damage.
To date, no studies have examined the combined effects of hypohydration and resistance exercise on the release of serum CK and Mb post-exercise. Based on previous research on muscle damage and fluid replacement during exercise, we hypothesized that resistance exercise and hypohydration in concert might increase the circulating concentrations of CK and Mb. Therefore, the purpose of this study was to examine the effects of hydration state on these indices of muscle damage following high-intensity resistance exercise performed by resistance-trained men.
Experimental Approach to the Problem
To study the effects of hydration and resistance exercise on muscle damage, subjects completed 3 identical resistance exercise bouts in different hydration states: euhydrated (HY0), hypohydrated by ∼2.5% body mass (HY2.5), and hypohydrated by ∼5% body mass (HY5). Hydration state was manipulated 1 day before testing by controlled water deprivation, exercise-heat stress, and fluid intake. Trial order was randomized and completed in ambient environmental conditions (21°C). Muscle damage markers were measured immediately preceding exercise (PRE), and immediately post- (IP), 1 hour post- (+1H), 2 hours post- (+2H), 24 hours post- (+24H), and 48 hours post-exercise (+48H). This study was part of a larger investigation and as such imposed some restrictions on the study design.
Seven healthy, resistance-trained men (age, 23 ± 4 years; height, 1.79 ± 0.58 m; body mass, 87.8 ± 6.8 kg; body fat, 11.5 ± 5.2%; back squat 1 repetition maximum [1RM] = 152 ± 20 kg) volunteered for this study. Before any familiarization or testing sessions, each subject was informed of the risks of the research and completed written informed consent approved by the University Institutional Review Board. Subjects were paid for their participation in the study.
Two preliminary testing sessions, on nonconsecutive days, occurred at least 1 week before the start of experimental testing. Subjects were weighed on several occasions in a postabsorptive, euhydrated state to establish baseline weight. A urine sample was analyzed for osmolality (Uosm) by freezing point depression (Model 3250, Advanced Instruments, Inc., Norwood, MA) and urine specific gravity (Usg) by refractometry (A300CL-E01, Atago, Tokyo, Japan) to document euhydration on all preliminary days; Usg ≤1.020 was defined as euhydration (1).
During the first familiarization session, the subject's 1RM squat was determined using a modified Smith Machine (LifeFitness, Schiller Park, IL). The subjects warmed up on a cycle ergometer (Model 818E; Monark, Stockholm, Sweden) for 10 minutes followed by self-determined active and passive stretching. Next, the subjects completed 1 set of 5-10 squats at approximately 50% of their estimated 1RM, followed by rest and a second warm-up set of 3-5 repetitions at ∼75% 1RM. One repetition maximum was established with 1 repetition of increasing weight with a minimum of 3 minutes of rest between attempts until a maximum weight was correctly lifted. A 1RM weight was achieved within 3-5 attempts, and squat depth was achieved when the femur was parallel to the ground (23).
On the second familiarization day, the subjects completed a high-intensity resistance exercise challenge (REC). The REC consisted of 3 sets of the parallel back squat at 80% of the subject's predetermined 1RM with 2 minutes of rest between sets. The subjects attempted to complete 10 repetitions per set. If a subject was unable to complete 10 repetitions, he stopped at exhaustion, but still attempted to complete all remaining sets. The total number of repetitions completed during these 6 sets served as the standard for all subsequent experimental REC testing.
The subjects were instructed to maintain their normal diet and training throughout the study. To standardize each trial, each subject provided dietary and exercise logs for 36 hours before and including the first testing day. The diet and exercise records were replicated for all experimental trials. Additionally, the subjects refrained from alcohol or other exercise stimuli for 36 hours before the start of testing.
Each experimental trial lasted 4 days and included a dehydration day, an exercise day, and 24- and 48-hour post-exercise evaluations.
On the morning of the first day of the experimental trial (DHY), the subjects arrived after fasting for 12 hours and were euhydrated for a baseline blood draw, a urine sample, and a baseline body mass. The subjects were then instructed to consume low-moisture foods and refrain from drinking for the remainder of the day.
The subjects returned to the laboratory during the afternoon of DHY for the dehydration protocol. For this, the subjects walked on a motorized treadmill at 3.4 mi·h−1 and a 2% incline in an environmental chamber (37°C, 40%-50% relative humidity [Model 2000, Minus Eleven, Inc., Malden, MA]). Nude body weight, heart rate (Vantage, Polar Electro, Woodbury, NY), rectal temperature (thermistor information), and rating of perceived exertion (3) were monitored at 20-minute increments. The subjects walked until they (a) had lost 5% of their morning body mass, (b) met preset safety criteria, (c) displayed signs or symptoms of an exercise-induced heat illness, or (d) requested to stop due to exercise fatigue.
After dehydration, the subjects were comfortably seated and given fluids, half orally and half intravenously (1 L·h−1 via each route) to return them to the appropriate hydration status (HY0, HY2.5, HY5). The oral fluid was a noncaloric, flavored, electrolyte beverage, and the intravenous fluid was normal saline (0.9% NaCl).
Following fluid replacement, the subjects consumed a high-calorie, carbohydrate (CHO)-rich meal (13 kcal·kg−1 body weight and 2.25 g CHO·kg−1 body weight; Classic Hand-Tossed Cheese Pizza, Domino's Pizza, Ann Arbor, MI). After this, the subjects were instructed not to eat, drink, or exercise before returning to the lab the following morning.
The subjects returned to the laboratory early the next morning. Upon arrival, they were weighed and provided a urine sample to verify hydration state. A 20-gauge Teflon cannula was inserted in an antecubital vein kept patent with normal saline and heparin (9:1).
After warming up, the subjects completed the REC squat protocol and then rested in a comfortable chair for 2 hours after exercise. Blood draws were take PRE, IP, +1H, +2H. After the +1H blood draw, the subjects were permitted to eat and drink ad libitum.
Days 3 and 4
The subjects reported to the laboratory in the morning of the 2 days following the exercise protocol (+24H, +48H); blood samples were taken with a 20-gauge butterfly needle. The subjects refrained from exercise until after their second evaluation day blood draw. To ensure rehydration, they were asked to consume extra fluids these 2 days.
Blood samples were drawn into plain or ethylenediamine tetraacetic acid- treated tubes (Vacutainer; Becton Dickinson, Franklin Lakes, NJ) at DHY, PRE, IP, +1H, +2H, +24H, and +48H. The hematocrit was determined in triplicate using a microcapillary technique, and hemoglobin (Hb) was measured in duplicate (Hb 201+; HemoCue, Ängelholm, Sweden) to determine the percentage of change in plasma volume (10). The remaining whole blood was centrifuged at 1500g for 15 minutes at 4°C. An aliquot of plasma was immediately assessed in duplicate for osmolality (Posm) by freezing point depression (3DII; Advanced Digimatic, Needham Heights, MA). Serum was aliquoted and frozen(−80°C) for subsequent duplicate analysis of lactate (HLa) using an enzymatic technique (Model 2300, Yellow Springs Inc., Yellow Springs, OH), Mb (Diagnostic Automation Inc, Calabasas, CA), and CK (Diagnostic Chemicals Ltd., Oxford, CT) as determined at 340 nm on a spectrophotometer (Biomate; ThermoSpectronic, Rochester, NY). Intra-assay coefficients of variation for HLa, Mb, and CK were 0.6%, 8.3%, and 1.4%, respectively.
Data are presented as mean ± SE. Data were analyzed with a repeated-measures (treatment × time) analysis of variance. When appropriate, Fisher's post hoc test was used to determine specific pairwise differences. Significance was set at p ≤ 0.05.
Hydration indices (body mass, Usg, Uosm, Posm) verified achievement of appropriate hydration states for each exercise trial. Subjects' body weight decreased (DHY to PRE) 0.2 ± 0.1%, 2.4 ± 0.1%, and 4.8 ± 0.1% for the HY0, HY2.5, and HY5 trials, respectively. Baseline Usg was <1.020 for all trials. Hypohydration followed by rehydration caused significantly greater Usg in the HY2.5 (1.025 ± 0.001) and HY5 (1.027 ± 0.001) trials than in HY0 (1.019 ± 0.001), and these significant differences were maintained overnight to PRE (HY0 = 1.020 ± 0.001, HY2.5 = 1.026 ± 0.001, HY5 = 1.027 ± 0.001). No differences existed between HY2.5 and HY5.
Baseline Uosm was <800 mOsm·kg−1 for all trials. Similar to Usg, hypohydration following rehydration caused significantly greater Uosm during HY trials (HY2.5 = 926 ± 16, HY5 = 972 ± 10 mOsm·kg−1) than HY0 (780 ± 10 mOsm·kg−1), and these significant differences persisted overnight to PRE (HY0 = 887 ± 15, HY2.5 = 1031 ± 13, HY5 = 1067 ± 14 mOsm·kg−1). No differences occurred between HY2.5 and HY5. Table 1 shows the Δ Posm and percentage of change in plasma volume for the exercise trials. Results did not change when selected variables were corrected for plasma volume shift; therefore, data are presented as uncorrected values.
HLa values (Table 2) were significantly elevated post-exercise, but did not differ among trials.
Figure 1 displays the Mb response to hypohydration and resistance exercise. At +1H and +2H, Mb values were significantly elevated above corresponding PRE values for all trials. Mb was not significantly different among trials. No significant differences in CK were noted over time or in response to the hydration state (Figure 2). Total work completed between trials was the same (Figure 3), with subtle differences attributed to squat depth.
Effect size (ES) was calculated for Mb and CK measures. Mb ES was large between PRE and +1H and +2H values for all trials (Table 3). CK ES was large between PRE and +24H (Table 3). The large ES corresponds to significant changes in Mb and CK concentrations. The scale for determining the magnitude of ES was a modified Cohen scale (29) that better reflects data from strength and conditioning research.
In opposition to our hypothesis, the main findings of this investigation indicate that muscle damage, assessed by Mb and CK measures, in response to strenuous exercise was not affected by mild to moderate levels of hypohydration in resistance-trained subjects. Athletes may damage muscle tissue during the course of training and competition (18,20). Further, many of those athletes do not adequately replace fluid lost during exercise (15,21,24). Our results suggest that regardless of hydration state, athletes did not incur additional muscle damage in response to the REC.
Previous studies examined the role of fluid ingestion on muscle damage (6,32); however, those studies did not examine the role of hydration state per se on muscle damage. Because indicators of postexercise hydration state were not provided in those studies, it was not possible to speculate regarding the degree of dehydration that those subjects experienced, if at all, or how this may have altered the Mb and CK responses. In the present study, subjects commenced exercise in both euhydrated and 2 different hypohydrated states. The degree of muscle damage sustained was not significantly different among trials. Therefore, it appeared that hypohydration did not contribute to the resultant muscle damage from the resistance exercise bout, although the data presented themselves in a stepwise manner consistent with hydration status.
Even though the exercise intensity was sufficient to stimulate increased serum Mb concentrations at +1H and +2H, CK concentrations remained stable. An exercise bout sufficient to stimulate Mb release may not be sufficient to stimulate CK release (12,14,30), especially in trained lifters (5,9,22); moreover, CK release alone may not represent muscle damage. Normally, CK concentrations peak 4-6 days post-exercise (5,8,34). Since this study was part of a larger investigation, subjects were required to exercise between trials to maintain training status. Therefore, CK sampling beyond 48 hours post-exercise was not possible, but increases are usually noted by this time (34). Figure 2 shows that CK concentrations, although not significantly elevated, peaked at +24H. Despite the high intensity of the REC, CK concentrations remained within physiologically normal resting values at all time points (36). Most studies found significant increases in CK concentrations after similarly demanding exercise protocols, regardless of training status (9,11,34). Clarkson et al. (4) found that the degree of muscle damage incurred in individuals was determined by genetic polymorphisms in myosin light chain kinase. In the present study, polymorphisms associated with increased Mb and CK post-exercise were not analyzed, which suggests that genetics, in addition to training status, may play an important role in the degree of sustained muscle damage.
No significant difference between trials existed for Mb or CK concentrations despite significant alterations in hydration state. In one of the few studies to examine the role of hydration and muscle damage, Clarkson et al. (6) used an isolated muscle damage design and found fluid ingestion necessary to maintain stable CK. Similarly, after +1H, subjects in this study drank ad libitum and increased fluid ingestion until they returned to a euhydrated state. Fluid ingestion may have affected serum muscle protein concentrations, indicated by decreased +2H serum Mb concentrations and no change in serum CK concentrations.
Muscle damage occurs through both metabolic and mechanical mechanisms (14). A possible metabolic mechanism for muscle damage in this study could have been increased glycogenolysis due to decreased fluid intake in the HY2.5 and HY5 trials, which created a greater physiological stress on muscle (13,17). Mechanical mechanisms likely created the largest contribution to the incurred muscle damage. It was apparent that eccentric contractions in the REC induced muscle damage sufficient to release Mb, but, due to the training status of the subjects, the stimulus was not great enough to sufficiently alter the membrane permeability to release the relatively large (80 kd) CK molecule. As cell membranes are composed of an anhydrous phospholipid bilayer (33), it was unlikely that the structural integrity was compromised by either dehydration or hypohydration. As a result, the induced muscle damage (i.e., elevated Mb concentrations) was attributed to the squat protocol alone and not by, or in conjunction with, the hydration state. It was highly unlikely that the combination of heat stress and exercise for the dehydration protocol on the day before the REC had an impact on muscle protein concentrations as Greiwe et al. (16) determined that heat stress and dehydration did not result in muscle damage.
In conclusion, hypohydration, although deleterious to performance, does not affect the magnitude of muscle damage consequent to resistance exercise in trained weight lifters. Further information may be gained by investigating the effects of hypohydration on resistance exercise-induced muscle damage on identified genetic responders to muscle damage in both trained and untrained weight lifters.
Exercising subjects voluntarily replace only about 70% of their net water loss during exercise (15,21), which suggests that many athletes may be chronically hypohydrated (2%-3%). In the present study, hypohydration did not significantly alter the degree of muscle damage sustained during an acute bout of resistance exercise. Importantly, circulating CK remained within normal levels after high-intensity resistance exercise for all 3 trials; however, the data did present themselves in a stepwise manner consistent with the degree of hypohydration. The resultant muscle damage from the REC is similar to values seen in experienced weight lifters in various sports (Table 4); values represent the highest postexercise measure of Mb and CK in each study.
Hypohydration at moderate levels (1%-5%) has consistently affected strength performance (31,35), core temperature (2,27), and hormonal profile (19,25,26). We advise athletes to initiate exercise in a euhydrated state to maximize the endogenous hormonal, mechanical, and metabolic benefits of resistance exercise training and to minimize the potential for muscle damage.
The authors thank the dedicated group of men who served as subjects for this demanding research project. Additionally, they acknowledge the invaluable technical support of Disa Hatfield, Barry Spiering, and Jakob Vingren.
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