In this study, 30 male subjects were primarily selected from student volunteers attending J University. The 30 subjects then had to undergo a physical examination to determine whether they had metabolic anomalies, cardiovascular anomalies, or any disorder associated with muscle as well as to ascertain whether they had ever used muscle enhancers. Students who were found to have diseases or to have taken muscle enhancers were excluded from the experiment. Before entering this experiment, the height, the weight, and the makeup of the subjects were measured to select the final 26 through the process to minimize errors. This was to minimize potential physical differences among the individual subjects in the clinical trial. All the process also received approval by the ethics committee. Thirteen participants were randomly assigned to the BCAA intake group and the others were assigned to the placebo group. All the subjects were educated to understand the behavior that could affect the experiment and submitted the experimental agreement. Physical characteristics of the subjects are shown in the <Table 1>.

In this study, the subjects rode on the bicycle ergometer (Corival, Monark Inc., USA) and started gradual maximum exercise after having taken BCAAs, which was dissolved in bottled water, and having drunken placebos (bottled water). Dependent variables were measured five times; before ingesting BCAAs and placebos; 10 minutes before exercise; 30 minutes into exercise; immediately after exercise; 30 minutes after exercise. Then the impact of the variables, which are related to fatigue substances, muscle damage substances, and energy metabolism substances, was analyzed. Preliminary experiments were conducted to all the subjects one week before the experiment. Drinking, smoking, and caffeine intake were banned three days before the experiment. All of the research was conducted at the same time every day in order to minimize the effects of variables including the experimental time, conditions, and the subjects’ circadian rhythm. Before the experiment, the researchers randomly determined the order in advance and proceeded with every experiment in that order. BCAAs and placebos were taken in accordance with a double-blind trial. The experiment was performed with an interval of one week to minimize the effects of previous exercise.

The test methods to analyze fatigue substances include a specific vehicle of EDTA-2Na with 8% Hypochlorite acid4 (HCIO4), 1% EDTA and 1% Ascorbic acid as a phosphorous acid at a pH of 3.5 in order to remove protein in Serotonin (5-HT). The study collected blood samples to agitate them in the centrifuge (HANIL Centrifuge MF_80) at a rate of 12,000 rpm for 20 minutes, and moved only the necessary amounts of them to the sample injection tube to measure them based on a L-8200 type of HPL (high performance liquid chromatography). To measure ammonia, this study used a spectrophotometer (CL-750) to observe Berthelot responses, and employed the spectrophotometer to measure lactate in the blood based on an enzyme method. To analyze CK, a muscle damage substance, the study used vacuum tubes for blood test to collect blood samples, which were then stirred at rates of 2500-3000 rpm for 15-20 minutes. The necessary amounts of bloods for measurement were once again extracted to analyze them with a dry biochemical analyzer (Kodak EKTACHEM DTSC Ⅱ, USA). To measure LDH, 2.70ml of Trisbuffer (57.5mmol/l), 0.1ml of NADH solution and 0.1ml extracted tissues were mixed evenly in a cuvette with a diameter of 1cm, and were then warmed up in a water at 30℃ for 10-20 minutes. The reactants was added with 0.2ml of pyruvate solution and evenly stirred before being measured with the dry bio-chemical analyzer (Kodak EKTACHEM DTSC Ⅱ) equipped with a heating device at a rate of 340nm at intervals of half or 1 minute for 3-6 minutes. To determine FFA, the study an energy metabolism substance, the study collected blood samples with the vacuum tube equipped with a clot activator designed to precipitate fibrinogen, agitated them in a centrifuge at rates of 2,500-3,000 rpm for 15-20 minutes, added 50 µl STD solution to 0.5 ml extracted blood serums to mix them a SICDIA NEFAZYME test reagent, and finally used Hitachi 7150 (Hitachi, Japan) to measure FFA after putting them in a cold water for 5 minutes. To measure glucose, the study agitated the collected blood samples in a centrifuge at rates of 2,500-3,000 rpm for 15-20 minutes and analyzed the supernatant liquid with a dry bio chemical analyzer (Kodak EKTACHEM DTSC Ⅱ, USA).

The study measured the mean and standard deviation of each variable by using the SPSS (ver 17.0). The study conducted the repeated ANCOVA with ‘before the intake of BCAA’ set up as a common variant to verify mean differences between groups and times. After having verified the gradients of the data prior to the test, the study found that the gradients of the data were all identical. In addition, in case that a significant difference happened, the study carried out a post verification test based on the Duncan method. The analysis on the correlation between the variables within each subject group was done with the Pearson correlation method.

The BCAA is the most easily oxidized type of amino acid in the skeletal muscles and serves as an important energy substrate when carbohydrate becomes exhausted after a prolonged exercise, as the contribution rate of fat as an energy source increases [21]. Serotonin is known to be involved in body temperature, blood pressure, internal secretion, appetite, sexual behavior, exercise, aggression, and pains [10] and adjust various biological functions such as memory and cognition [1]. According to the previous studies on serotonin associated with exercise, the results of the measurement for the activation level of serotonin at the raphe at the rate of 5-HIAA/5-HT showed that an animal group which worked out up to 50% of the maximum exercise capacity showed a significantly higher activation percentage compared with the other animal group which exercised up to the maximum exercise capacity [5]. Moreover, an increase in serotonin was reported in white rats after exercise, according to the study on the effects of intensity of treadmill exercise and duration of mild-treadmill exercise on serotonin activity in the brain of rats [11]. This study measured the levels of serotonin, ammonia and lactate, with an aim to assess the effect of the intake of BCAA on fatigue substances, and found that the serotonin in a group that consumed BCAA was decreased at 10 min before exercise and 30 min into exercise but slightly increased at immediately after exercise before being dropped again at 30 min after exercise. In the placebo group, the level of serotonin was increased at 10 min before exercise but started to be decreased at 30 min into exercise being escalating again at immediately after exercise and 30 min after exercise. In regard to the variances between the subject groups, the placebo group showed a statistically significant difference; it indicated a higher serotonin concentration at 10 min before exercise and 30 min after exercise than the BCAA intake group, while the serotonin level in the BCAA intake group was lower than the placebo group at all test times, which is identical to the results of the study by Lee Han, et al. [12]. The study results by Wagenmakers, et al. [20]. which administered the BCAA into those patients who suffered from McArdle's disease and had them to ride a bicycle ergometer, suggested that the ammonia concentration after exercise had increased by about 10 times compared to that in a stable condition. MacLean and Graham[15] administered the BCAA into healthy people with a normal level of glycogen concentration and had them ride a bicycle ergometer. The study results showed that the BCAA intake group indicated a significantly higher ammonia concentration in the blood after exercise than the non-BCAA intake group. In this study, the ammonia levels in the both groups were increased at 10 min before exercise, 30 min into exercise and immediately after exercise, but were decreased by a statistically significant degree at 30 min after exercise. In regard to the variances between the groups, the placebo group indicated a statistically meaningful disparity with a higher concentration of ammonia at 30 min after exercise compared with that of the BCAA intake group, while the BCAA intake group recorded a higher concentration of ammonia at nearly all test times, which is similar to the results of the studies by MacLean and Graham [15]. The ammonia concentration was increased during exercise but was reduced to a stable level during recovery. It is presumed that the ratio of fat as an energy source during endurance exercise rose due to the activation of fat oxidization, while that of the protein as an energy source was relatively dwindled, reducing the concentration of ammonia in the blood. Lactate was formed as a result of anaerobic metabolism of glucose during exercise, and is the important barometer in determining the limitation factors of muscle fatigue and muscle activity. In this study, the lactate levels of the two groups, respectively, showed a statistically significant increase at 30 min into exercise. The lactate levels in the BCAA intake group indicated a statistically meaningful decrease at immediately after exercise and returned to a stable level at 30 min after exercise. In regard to the variances between the groups, the BCAA intake group showed a lower concentration of lactate at immediately after exercise than the placebo group. The reason why the lactate level in the blood reached the peak at the end of exercise is because the amounts of oxygen uptake and that oxygen which transported to the muscle tissues were reduced as the exercise proceeds, which in turn caused the accumulation of lactate in the body by hindering pyruvate from entering the TCA cycle efficiently and transforming it into lactate.

The concentrations of CK and LDH, both of which play an important role in adjusting the energy metabolism required for muscle activity, can be used as a crucial indicator in determining the levels of muscle damage and body robustness [19]. Moreover, LDH, an indicator of muscle damage, is known to play a crucial role in formation and transformation of lactate in muscle cells, among muscle activities. As an oxidization and reduction enzyme, it converts pyruvic acid into lactate in the process [17]. Liu, et al. [14] reported that the CK and LDH levels in the group who consumed the BCAA for 5 weeks were measured after endurance and elastic band exercises. Greer, et al. [7] reported that the BCAA intake group showed lower concentrations of CK and LDH after they rode a bicycle ergometer at a rate of 50% of maximum oxygen uptake capacity for a prolonged time than otherwise. Matsumoto, et al. [16] reported that a group of 12 long distance runners who consumed beverages containing the BCAA while undergoing a three-day intensive training showed lower concentrations of both CK and LDH compared with the placebo group. The study indicated that the CK levels of both groups were increased at 30 min into exercise but reduced to lower than pre-exercise level at immediately after exercise and 30 min after exercise. The CK concentration change patterns depending on test times of both groups showed similar results, but the placebo group had a higher concentration of CK than the BCAA intake group at all test times. The LDH levels of both groups were higher at 30 min into exercise but were decreased at immediately after exercise and 30 min after exercise, compared with those before the intake of the BCAA. The LDH concentration change patterns depending on test times showed the same results. However, the placebo group had a higher concentration of LDH than the BCAA intake group. If all the above mentioned study results are taken into account, it is presumed that an reduction of concentration of muscle damage substances such as CK and LDH can help contribute to enhancing exercise performance.

Exercise can increase the influx of glucose into cytoplasm by rapidly stimulating the glucose transport activity in a muscular fibrous coat [6]. The concentration levels of plasma glucose and FFA during exercise have a strong correlation. Meanwhile, Gualano, et al. [8] reported that a group which consumed 300mg of BCAA per unit weight on a daily basis showed a higher concentration of glucose after an exhaustive exercise. However, this study observed that the glucose levels in the BCAA intake group were slightly increased at 10 min before exercise and 30 min into exercise and were decreased at immediately after exercise and 30 min after exercise, in comparison with those before the intake. When it comes to the variances between the groups, the placebo group showed a statistically meaningful disparity with a higher concentration of glucose at 30 min after exercise compared to the BCAA intake group. Although the glucose concentration change patterns depending on test times showed similar results in both of the groups, the placebo group showed a higher concentration of glucose at all test times than the BCAA intake group. The concentration levels of FFA in the BCAA intake group showed a statistically meaningful increase at 10 min before exercise, 30 min into exercise, immediately after exercise and 30 min after exercise. The FFA concentration change patterns depending on test times showed that the BCAA intake group showed a lower concentration at 10 min before exercise and 30 min into exercise, in comparison with the placebo group, but indicated a higher concentration of FFA at immediately after exercise and 30 min after exercise than the BCAA intake group. Moreover, the BCAA intake group showed a higher concentration of FFA at immediately after exercise especially after one hour of exhaustive exercise, which is identical to the results of the studies by Zhang, et al. [22] and by Gualano, et al. [8]. It is presumed that this is due to the fact that the glucose is used as an energy source during exercise but is preplaced with FFA at immediately after exercise, for example, in case of more than one hour of exhaustive exercise. If all the above mentioned study results are taken into consideration, the BCAA intake can lower the concentration of serotonin, a central fatigue substance, which can in turn reduce the concentrations of muscle damage substances such as CK and LDH. That is to say that the less secretion of fatigue substances means the less secretion of those enzymes that can cause muscle damages, and subsequently can enhance exercise performance. Therefore, the intake of the BCAA is presumed to contribute to reducing the concentrations of muscle damage substances such as CK and LDH and enhancing the exercise performance. Meanwhile, an increase in the ammonia concentration can deteriorate exercise performance, while a reduced secretion of FFA means a decreased secretion of LDH. In addition, the study confirmed that glucose is used as an energy source during exercise but is replaced with FFA at immediately after exercise, for example, in case of more than one hour of exhaustive exercise, which in turn can improve exercise performance and plays an effective role in preventing muscle damages. However, as this study was based on the results of those previous studies which suggested that the intake of the BCAA before exercise is the most effective in enhancing exercise performance, more follow-up studies should be conducted in consideration of other variables such as gender, age, disease and nutrition status, and also more studies on the effect of the BCAA administration conditions (dosage, duration, administration type) on exercise types, intensity and times need to be carried out in the future.