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Hormonal Responses to Resistance Exercise after Ingestion of Carnosine and Anserine

Goto, Kazushige; Maemura, Hirohiko; Takamatsu, Kaoru; Ishii, Naokata

The Journal of Strength & Conditioning Research: February 2011 - Volume 25 - Issue 2 - p 398-405
doi: 10.1519/JSC.0b013e3181bac43c
Original Research

Goto, K, Maemura, H, Takamatsu, K, and Ishii, N. Hormonal responses to resistance exercise after ingestion of carnosine and anserine. J Strength Cond Res 25(2): 398-405, 2011-Intramuscular carnosine buffers protons (H+) in skeletal muscle. We examined the effects of supplementation with chicken breast meat extract (CBEX) containing carnosine and anserine on hormonal responses to resistance exercise. Twenty-two men were assigned to a CBEX drink group (CBEX containing total 2 g of carnosine and anserine) (n = 14) or a placebo drink group (n = 8). The subjects ingested the prescribed drink (100 mL) twice daily for 30 days without physical training. Before and after the supplementation period, the subjects completed 5 sets of bilateral knee extension exercises (with a 90-s rest between sets). The magnitude of the increase in exercise-induced free testosterone did not change significantly after supplementation in either group. The blood lactate response to exercise was attenuated after supplementation in both groups (p < 0.05). In the CBEX group, the plasma epinephrine and norepinephrine concentrations after exercise were significantly lower after supplementation (p < 0.05). The serum growth hormone response to exercise was also reduced in the CBEX group after supplementation (delta value: 5.4 ± 1.9 ng/mL [pre] vs. 1.6 ± 0.5 ng/mL [post], p = 0.05). No significant differences in exercise-induced strength reduction (fatigue index) were observed in the 2 groups after supplementation. These results suggest that short-term supplementation with CBEX attenuates the exercise-induced epinephrine, norepinephrine, and growth hormone responses.

1Faculty of Sport Sciences, Waseda University, Tokorozawa, Saitama, Japan; 2Faculty of Health and Sport Sciences, International Pacific University, Okayama, Okayama, Japan; 3R & D Center, Nippon Meat Packers, Inc., Tsukuba, Ibaraki, Japan; 4Faculty of Health and Sport Sciences, Ryutsu keizai University, Ryugasaki, Ibaraki, Japan; and 5Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Tokyo, Japan

Address correspondence to Dr. Kazushige Goto,

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Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-l-methyl-L-histidine) are histidine-containing dipeptides present in the brain, heart, liver, and skeletal muscles (4). Intramuscular carnosine has several physiologic functions, including pH buffering (12), antioxidant activity (2), the regulation of Ca2+ sensitivity in excitation-contraction coupling (23), and the protection of proteins against glycation by acting as a sacrificial peptide (16). Human muscle does not contain anserine (26), but 4 weeks of β-alanine supplementation increases the intramuscular carnosine concentration by approximately 60% (13,15), raises the ventilatory threshold during submaximal-intensity exercise (34), and increases the total work volume during intensive cycling (15). These results suggest that muscle carnosine loading after β-alanine supplementation enhances the H+-buffering capacity of skeletal muscle, thereby improving exercise performance.

Resistance exercise is a potent stimulus for endocrine activity, such as the increased release of anabolic (e.g., growth hormone [GH], testosterone) and catabolic hormone (e.g., cortisol) concentrations (9,22). Muscle growth can take place even in the absence of marked increases in circulating anabolic hormone concentrations (39), but exercise-induced anabolic hormone secretions are believed to play a role in muscular hypertrophy (22). The exercise-induced acute increase in GH correlates positively with the magnitude of muscular hypertrophy after training (28). Madarame et al. (24) have also recently shown that resistance exercise with blood flow restriction for leg muscles stimulates acute increases in plasma noradrenaline concentrations and augments an increase in the cross-sectional areas of arm muscles subjected to normal resistance training after 10 weeks. These findings suggest that a knowledge of the acute changes in anabolic hormones and catecholamines should be useful in prescribing optimal exercise guidelines.

A growing body of evidence strongly suggests that the GH response to resistance exercise is mainly attributable to metabolite accumulation and the subsequent metaboreflex by way of muscle chemoreceptor stimulation through type III and IV afferents (8,31,32). In fact, the increased intramuscular metabolite accumulation caused by moderate vascular occlusion markedly enhances the GH response to resistance exercise (31,32,37). In contrast, enhanced metabolite accumulation within muscles does not affect the testosterone response (6,8,32). A previous study using a sensory nervous blockade indicated that afferent nervous activity is important in the regulations of the resistance-exercise-induced catecholamine responses (21). Conversely, the enhanced motor center activity (central command) induced by a partial neuromuscular blockade elicited an adrenocorticotropic hormone (ACTH) response to exercise (19), suggesting the role for motor centers activity in the regulations of ACTH-cortisol secretions. It appears that the relative contribution of intramuscular metabolite accumulation (or central command) to exercise-induced hormonal responses is dependent on the types of hormones involved.

Although the effects of β-alanine supplementation have already been documented, recent studies (29,33) have focused on the effect of dietary chicken breast extract (CBEX) containing carnosine and anserine on intramuscular carnosine concentration. In adults, carnosine is rarely found in the blood because of the rapid action of the carnosine-degrading enzyme, carnosinase (3,27). However, it has been assumed that the circulating β-alanine and histidine derived from consumed CBEX can be absorbed into skeletal muscles, leading to the resynthesis of carnosine (1). We previously reported that short-term CBEX supplementation increases intramuscular carnosine concentration in both untrained subjects (approximately 28%) (33) and rats (approximately 15%) (29). Enhanced intramuscular carnosine affects the H+-buffering capacity of muscles during resistance exercise (5). Thus, it is possible that carnosine supplementation alters the resistance exercise-induced hormonal responses. This study was designed to examine the effects of short-term carnosine and anserine supplementation on the hormonal and metabolic responses to resistance exercise. We hypothesized that 30 days of carnosine and anserine supplementation would attenuate the resistance exercise-induced GH and catecholamine responses but would not affect testosterone and cortisol responses.

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Experimental Approach to the Problem

This study was conducted using a blind, placebo-controlled design. After the measurement of 1 repetition maximum (1RM) for the bilateral knee extension exercise, each subject was assigned to either the CBEX group (n = 14) or the placebo supplement group (placebo group, n = 8). The CBEX group had larger number of subjects because we were especially interested in determining whether with the consumption of the CBEX drink alters the hormonal responses after resistance exercise. The CBEX drink contained carnosine and anserine (35). The subjects in each group consumed the CBEX drink (CBEX group) or the placebo drink (placebo group) daily for 30 days. During this period, they were instructed to drink 2 bottles each day (1 in the morning, 1 in the afternoon). The subjects were also asked to maintain their regular dietary patterns and abstain from exercise training and other nutritional supplements. Before and after the 30-day supplementation period, the subjects completed 5 sets of bilateral knee extension exercises to determine their exercise-induced hormonal responses. The subjects did not consume the experimental drink on the exercise day. When the subjects were unable to perform the exercise test immediately after the 30 days of supplementation, they were asked to continue consuming the drink as scheduled until the evening before the chosen exercise day.

Dietary records were collected in 2 periods for 3 consecutive days before and after the supplementation period. Commercial software (Dietary Consulting Room, Olympus Co., Ltd, Japan) was used to analyze meal contents to determine nutrient intake.

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Twenty-two healthy men (mean age ± SE: 25 ± 1 yr; height, 174 ± 1 cm; body mass, 68 ± 2 kg) participated in this study. None of the subjects were involved in any regular training program at the start of the experiment. Subjects were informed about the experimental procedure and the purpose of this study. Their written informed consent was subsequently obtained. This study was approved by the local research ethics committee.

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Preparation of CBEX and Placebo Drinks

The CBEX drink is difficult to ingest directly because of its strong flavor, so we specifically manufactured a drink mixed with 20 g CBEX (containing total 2 g carnosine and anserine) per bottle (100 mL) using a method similar to that described in previous studies (25,33,35). The subjects consumed 2 bottles each day (total 4 g of carnosine and anserine) for 30 days. The amounts of carnosine and anserine administered were determined on the basis of a previous human study, which reported an increase in intramuscular carnosine concentration after 30 days of CBEX supplementation (33). The placebo drink was prepared with almost identical ingredients and a similar taste but without CBEX. Other than the chicken extract, no animal extract was used to prepare either the CBEX or placebo drinks. Relevant nutritional values are presented in Table 1.

Table 1

Table 1

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Resistance Exercise

Subjects visited the laboratory before the supplementation period to evaluate 1RM for the bilateral knee extension exercise using a weight-stack machine (Pro2SE, Life Fitness Co., Ltd., Tokyo, Japan). Before measuring 1RM, the subjects performed warm-up sets of 10 repetitions and stretching of the major muscle groups. The load was increased gradually until the subjects were unable to complete a lift. A rest of at least 2 minutes was taken between each trial to avoid fatigue. The subjects subsequently performed 3 sets of the exercise for familiarization.

Before and after the supplementation period, all subjects performed bilateral knee extension exercises from a seated position to determine their exercise-induced hormonal responses. Each exercise bout consisted of 5 sets, with 90-second rest periods between sets. The exercise intensity was set at 60% of 1RM in the first set. For sets 2 to 5, the load was generally reduced by 10% of 1RM after each set. The subjects were instructed to exercise with slow movement (2 s for lifting action, 2 s for lowering action) to maximize metabolite accumulation in working muscles. Before the supplementation period, they performed each set of exercises until exhaustion. We used relatively low-intensity exercise with slow movement because low-intensity exercise at a slow velocity causes marked increases in metabolite accumulation within the muscles, leading to the stimulation of anabolic and catabolic hormone secretions (10). The repetitions in the first set were 12 ± 1 for the CBEX group and 12 ± 1 for the placebo group. After the supplementation period, the subjects performed the exercise at the same intensity, with the same number of repetitions in each set as before the supplementation period. Therefore, we manipulated the number of repetitions in each set to match the work volume before and after the supplementation period. The range of motion in each set of exercises was from 90° to 0° (0° at full extension). The resistance exercise in each trial was performed at almost the same time of day to avoid diurnal variations in the metabolite and hormonal responses. Heart rate (HR) was monitored continuously (every 5 s) throughout the exercise session using a wireless HR monitor (Accurex Plus; Polar, Oy, Kempele, Finland).

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Blood Sampling and Analyses

After an overnight fast, the subjects arrived at the laboratory and rested for 30 minutes before the first blood collection. Venous blood samples were obtained from an antecubital vein before exercise, immediately after exercise, and 15 and 30 minutes after exercise to determine the metabolite and hormone concentrations. Blood samples for the measurements of hormones were kept on ice and were subsequently centrifuged at 3,000 rpm for 10 minutes to obtain the serum or plasma. The samples were stored at −85°C until analysis. The concentrations of blood metabolites and hormones were determined using the method described in a previous study (9). Concentrations of plasma epinephrine and norepinephrine were measured using high-performance liquid chromatography with kits (Tosoh Corp., Tokyo, Japan). The interassay and intra-assay coefficients of variation (CVs) were 0.2% and 2.6% for epinephrine and 0.4% and 3.9% for norepinephrine, respectively. The limit of detection was 6.0 pg/mL for epinephrine and norepinephrine. The serum GH concentration was measured using radioimmunoassay (RIA) with a kit (SRL, Ltd., Tokyo, Japan). The interassay and intra-assay CVs were 4.1% and 3.3%, respectively. The limit of detection for GH was 0.04 ng/mL. The serum free testosterone and cortisol concentrations were measured using an RIA with commercially available kits (Diagnostic Products Corp., USA; Immunotech, Inc., Marseille, France). The interassay and intra-assay CVs were 4.6% and 5.7% for free testosterone and 3.1% and 4.7% for cortisol, respectively. The limit of detection was 0.6 pg/mL for free testosterone and 1.0 μg/dL for cortisol. Blood lactate concentrations were determined immediately after the collection of the blood using an automatic lactate analyzer (YSI 1500 Sport; YSI, Inc., Yellow Springs, OH, USA).

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Measurements of Muscular Strength

The maximum isokinetic strength of the unilateral knee extension exercise was measured before and immediately after the resistance exercise to assess muscular fatigue. The exercise-induced strength loss (fatigue index, %) was calculated from the values obtained. The maximum isokinetic strength was measured using an isokinetic dynamometer (COMBIT; Minato Medical Science Co., Ltd., Tokyo, Japan). Each subject sat on a chair with the ankle of the right leg (dominant side) attached firmly to the lever of the dynamometer with a strap. The pivot of the lever was aligned accurately with the rotation axis of the knee joint. The requisite axial alignment of the joint and dynamometer axes was maintained during the movement. The maximum isokinetic strength was measured at 180°/s. Three repetitions with maximal effort were performed to determine the peak torque for joint angles ranging from 90° to 0°. The intraclass correlation coefficient for the measurements with this method was 0.95.

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Statistical Analyses

Data are expressed as means ± SE. A 2-factor (period [pre- and postsupplementation]) × time [before exercise and at 0, 15, and 30 min after exercise]) analysis of variance (ANOVA) with repeated measures was initially used. When ANOVA revealed a significant interaction or main effect, post hoc tests were performed to assess where the difference occurred. The delta values for the hormonal concentrations were defined as the actual differences between the baseline values and the maximum values after exercise. The delta values for the hormonal responses and fatigue index were compared between the pre- and postsupplementation periods using a paired t-test. p ≤ 0.05 was considered significant.

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Dietary Records

Daily nutrient compositions were calculated from obtained dietary records over 3 consecutive days. Total energy intake was not significantly different between the groups (presupplementation: 1,781 ± 100 kcal/day for CBEX, 1,725 ± 101 kcal/day for placebo; postsupplementation: 1,689 ± 92 kcal/day for CBEX, 1,698 ± 97 kcal/day for placebo). The nutrient compositions (i.e., protein, lipid, carbohydrate, salt) did not differ between the groups over the experimental period. No subjects reported discomfort or side effects after supplementation.

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Blood Lactate

Before supplementation, the blood lactate concentration increased significantly after the exercise in the CBEX (from 0.5 ± 0.1 to 4.7 ± 0.3 mmol/L) and placebo groups (from 0.6 ± 0.1 to 5.3 ± 0.6 mmol/L, p < 0.05). After supplementation, the lactate concentration increased after exercise in both groups (p < 0.05), but the responses were slightly but significantly lower than the responses before supplementation (interaction between period and time, p < 0.05 for both groups).

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Circulating Hormones

The plasma epinephrine concentration increased significantly after exercise in both groups (p < 0.05) (Figure 1). The epinephrine response to exercise was smaller in the CBEX group after supplementation (interaction between period and time, p < 0.05). In the CBEX group, the exercise-induced increase (Δ value) in epinephrine was significantly lower after supplementation than before supplementation (p < 0.05). In the placebo group, the resting epinephrine concentration was lower at postsupplementation than at presupplementation (p < 0.05), but the interaction effect was not significant after supplementation. No significant difference was observed in the exercise-induced increase in epinephrine before and after supplementation. The plasma norepinephrine concentration increased significantly after exercise in both groups. The norepinephrine response to exercise was only significantly smaller in the CBEX group after supplementation (interaction between period and time, p < 0.05).

Figure 1

Figure 1

Before supplementation, the serum GH concentration increased after exercise in both groups (Figure 2). The GH response to exercise tended to be smaller in the CBEX group after supplementation (interaction between period and time, p = 0.07). The value of the exercise-induced GH increase was also significantly lower after supplementation (1.6 ± 0.5 ng/mL) than before supplementation (5.4 ± 1.9 ng/mL, p ≤ 0.05). In the placebo group, the GH response to exercise was similar before and after supplementation.

Figure 2

Figure 2

In the CBEX group, the serum free testosterone concentration increased significantly after exercise (p < 0.05), but the response was similar before and after supplementation (Figure 3). The free testosterone response was also similar before and after supplementation in the placebo group. Before supplementation, the serum cortisol concentration increased significantly after exercise in both groups (p < 0.05). The cortisol response to exercise was smaller in both groups after supplementation (interaction, p < 0.05).

Figure 3

Figure 3

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Muscular Strength

Maximum isokinetic strength before exercise did not change over the experimental period in the CBEX group (presupplementation, 165.7 ± 7.9 Nm; postsupplementation, 169.8 ± 7.9 Nm) or the placebo group (presupplementation, 164.7 ± 13.1 Nm; postsupplementation, 160.1 ± 9.8 Nm). Before supplementation, the maximum isokinetic strength decreased drastically immediately after exercise in both groups (p < 0.05). After supplementation, no significant change in the fatigue index was observed in either group. The HR throughout the exercise session was similar before and after supplementation in both groups (CBEX, 113 ± 5 to 111 ± 5 beats/min; placebo, 123 ± 6 to 117 ± 7 beats/min).

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The 30-day supplementation with CBEX containing carnosine and anserine significantly attenuated epinephrine, norepinephrine, and GH responses to resistance exercise. We did not directly measure the intramuscular carnosine concentration, but our earlier studies with carnosine and anserine (CBEX) supplementation demonstrated increases in the intramuscular carnosine concentration in untrained subjects (33) and rats (29). On the basis of these findings, the intramuscular carnosine concentration would be expected to increase after the treatment period in the CBEX group in the present study. Intramuscular carnosine buffers H+ during intensive exercise (15). The enhanced muscle buffering capacity may alter the exercise-induced hormonal responses to resistance exercise.

Efferent signals (central command) and afferent signals from working muscles regulate the neuroendocrine and cardiovascular responses to exercise (18). However, the contribution of these factors to hormonal secretions appears to be dependent on the type of exercise. For instance, previous studies demonstrated that efferent signals were more important in regulating the exercise-induced catecholamine, GH (21), and ACTH response (19). On the other hand, the same research group also showed that an afferent nervous blockade attenuates catecholamine response to static resistance exercise (20), suggesting the importance of afferent signals. Furthermore, low-intensity resistance exercise with moderate vascular occlusion stimulates GH secretion (31,32,37). These findings suggest that metabolite accumulation within muscles is an important factor in activating catecholamines and GH releases after resistance exercise. As we expected, the catecholamine and GH responses to resistance exercise were significantly lower in the CBEX group after the treatment period. The lactate response was also smaller in the CBEX group after the treatment period. However, it is noteworthy that the lactate response after the treatment period was also impaired in the placebo group. The maximum strength and fatigue index did not change over the supplementation period, but familiarization (e.g., experience of intensive resistance exercise session during the presupplementation test) may partially explain the reduced hormonal responses in the CBEX group.

A recent study demonstrated that muscle caronosine loading after β-alanine supplementation attenuated fatigue during repeated bouts of knee extension exercise (5). Hoffman et al. (17) also showed that power output during squat exercise increased after 30 days of β-alanine supplementation. In contrast, with CBEX supplementation, the circulating β-alanine and the histidine derived from the consumed CBEX appeared to be taken into the skeletal muscle, resulting in the resynthesis of carnosine (1). We observed rapid increases in the blood β-alanine and histidine concentrations after the ingestion of the CBEX drink (36). In the present study, we expected that CBEX supplementation would impair exercise-induced strength reduction, but no beneficial effect of the CBEX supplementation on the fatigue index was observed.

The serum free testosterone concentration represents the amount of testosterone that is biologically available to cells because 98% of testosterone is bound to binding proteins (14). Resistance exercises result in increased serum free testosterone concentrations after exercise (6). The free testosterone concentration especially increases after intensive resistance exercise with moderate intensity and short rest periods between sets (muscular hypertrophy protocol) (9). Furthermore, a significant increase in the free testosterone concentration is observed during low-intensity resistance exercise when the exercise is conducted at slow velocity (tonic force output) (10). In the present study, supplementation with carnosine and anserine did not alter the free testosterone, either at rest or in response to exercise. These results are supported by findings that the magnitude of metabolic stress does not influence the acute testosterone responses to resistance exercise (6,8,32).

In addition to anabolic hormones, the cortisol response is important in muscle adaptations because the testosterone/cortisol ratio is implicated in the anabolic responses after resistance exercise (32). The difference in metabolic stress does not influence changes in cortisol during exercise (32). In the present study, the cortisol response to exercise was similar in the CBEX group and placebo group. Therefore, carnosine and anserine supplementation does not appear to affect the cortisol response.

The present study has several limitations. The first limitation is the lack of intramuscular data after supplementation. Unfortunately, we could not determine the intramuscular carnosine concentration. As described above, we used CBEX drinks with a daily total content of 4 g carnosine and anserine. In an earlier study using a similar CBEX drink, we demonstrated that 30 days of supplementation with 4.5 g or 1.5 g carnosine and anserine increased intramuscular carnosine concentration (33). We also used untrained subjects, who have lower carnosine levels (30), because there might be a ceiling effect in the response to carnosine supplementation, as with muscle creatine loading (11). Therefore, we speculate that intramuscular carnosine concentration increased sufficiently in the present CBEX group. Another limitation is the absence of blood pH data. The results of a previous study using the CBEX drink (containing 1.5 g carnosine and anserine) indicated that CBEX supplementation attenuates the reduction in blood pH during intensive exercise, without any significant change in the blood lactate response (35). Surprisingly, the lactate response to exercise decreased after the supplementation period in both the CBEX and placebo groups, suggesting that blood lactate concentrations do not play a major role in the attenuated catecholamine and GH responses in the CBEX group. We were unable to clarify the effects of CBEX supplementation on exercise-induced metabolic stress. The blood lactate concentration is widely accepted as an indicator of exercise-induced metabolic stress (8), but the blood pH response may reflect the intramuscular metabolic stress more directly. According to a previous study that demonstrated the role of the acid-base balance in GH secretion, blood hydrogen ion (pH) is closely related with the GH response to intensive exercise (7). Therefore, we assume that data for the blood pH response might be useful in clarifying the effect of CBEX on the metabolic stress and hormonal responses.

In conclusion, 30 days of CBEX supplementation with carnosine and anserine attenuated circulating epinephrine, norepinephrine, and GH after resistance exercise. In contrast, supplementation with CBEX did not influence the free testosterone response to resistance exercise.

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Practical Applications

From a practical perspective, the lower sympathetic activity during exercise (indicated by lower epinephrine and norepinephrine responses) after CBEX supplementation may assist untrained people who undertake resistance training (e.g., clinical populations or the elderly) because the rapid rise of blood pressure caused by resistance exercise should be considered in these people. In animal studies, carnosine administration suppressed renal sympathetic nerve activity and blood pressure (38).

Previous studies have shown that CBEX supplementation improves the time to exhaustion during fatiguing endurance exercise (25) and attenuates the decline in blood pH during repetitive maximal sprint exercise (35). However, our present findings did not demonstrate any beneficial reduction in resistance exercise-induced fatigue. The administration of CBEX reduced the catecholamines and GH responses after resistance exercise. Because exercise-induced increases in these hormones would be expected to play a role in promoting protein synthesis and fat metabolism after exercise, athletes and strength coaches should consider this when they use CBEX supplementation combined with resistance training.

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The authors are grateful to the subjects who participated in this study. The study was supported by a Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The experimental drink was provided by the Nippon Meat Packers, Inc., Japan. H. Maemura was a research associate of the company when the research was conducted. The results of the present study do not constitute endorsement of the product by the authors or the NSCA.

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buffering action; epinephrine; norepinephrine; growth hormone

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