Subjects
This study was approved by the Ethics Committee of Nippon Sport Science University (ID: 012-H50). All participants were informed verbally, as to the aim and the potential risks of the experiments and provided written informed consent before starting this study, which complied with the Declaration of Helsinki. A total of 18 males aged 19.2 ± 0.4 years, 64.9 ± 7.3 kg, 170.9 ± 5.6 cm tall, and 12.5 ± 3.4 % body fat, took part in the study. Subjects were recreationally active (moderate exercise 4–5 times a week) and considered apparently healthy without a history of disease or medication use. Subjects reported that they had not engaged in any intense training, including eccentric contractions, sufficient to cause significant muscle soreness in the 3 months prior to experiment.
Experimental procedure
The subjects started supplement ingestion one week before the exercise experiment, and continued it until 4 days after the exercise, i.e., the duration of the ingestion period was 12 days in total. On the day of exercise, after body composition, blood sampling, subjective muscle soreness (Visual Analogue Scale: VAS) of the biceps brachii of the non-dominant arm, and maximum isometric muscle strength of the elbow were measured, followed by eccentric contraction exercise of the same arm. Blood analysis, VAS, and maximum isometric muscle strength of elbow flexion were performed 1, 2, 3, and 5 days after exercise (Fig. 1). Vigorous exercise causing muscle soreness, icing, massage, and ingestion of other supplements were prohibited throughout the experimental period.
Supplements
The 18 subjects were randomly divided into 3 groups: groups for simultaneous ingestion of HMB and whey protein isolate (HMB + Whey group: n = 6, age: 19.0 ± 0.0 years, height: 168.6 ± 6.2 cm, weight: 60.7 ± 7.1 kg, body fat: 11.2 ± 3.3 %, lean body mass: 53.8 ± 5.5 kg), HMB alone (HMB group: n = 6, age: 19.3 ± 0.5 years, height: 170.6 ± 3.9 cm, weight: 67.3 ± 7.9 kg, body fat: 13.6 ± 3.7 %, lean body mass: 58.0 ± 5.1 kg), and whey protein isolate alone (Whey group: n = 6, age: 19.3 ± 0.5 years, height: 173.4 ± 6.2 cm, weight: 66.7 ± 6.2 kg, body fat: 12.7 ± 3.3 %, lean body mass: 58.1 ± 4.5 kg). The experiment was performed employing the double-blind method. For HMB, calcium-HMB capsules containing 1 g of HMB per capsule were ingested (Optimum Nutrition, Inc., USA): One capsule was ingested with water 3 times a day after breakfast, lunch, and supper (HMB: 3 g/day). For whey protein, whey protein isolate (ISO PRO, Bulk Sports, Japan) was ingested. A spoonful of whey protein (21 g, energy: 79 kcal, protein: 18.3 g, fat: 0.3 g, carbohydrate: 1.0 g, and sodium: 147 mg) mixed with 200 ml of water was ingested twice a day, after breakfast and supper (Whey protein isolate: 36.6 g/day). The ingredient percentage of essential amino acid and branched-amino acid in the whey protein isolate were 45.3 and 22.1 %, respectively.
Eccentric exercise
Eccentric exercise was performed using the Biodex System 3 (Biodex Medical Systems, Inc., USA). The subject sat on the measurement device and placed the upper arm on the pad so as to set the shoulder joint of the non-dominant arm at 45° flexion. After adjusting the rotation axes of the elbow joint and dynamometer to the same level, the chest and lumbar region, and upper arm of the subject were fixed with straps. The subject then grasped and adjusted the moment arm to a length giving no discomfort to the wrist joint at 90 and 180° of the elbow joint angles. In the eccentric exercise of the elbow, eccentric contraction was repeated 6 times with maximum effort as one set, and 7 sets were performed at 2-min intervals (42 eccentric contractions in total, angular velocity: 30°/sec, range of motion: 90–180°).
Measurement
Body mass and body composition were measured using a Tanita MC-190 multi-frequency body composition monitor (TANITA Co., Japan). Blood samples (~10 ml) were taken from the median antebrachial vein, and serum was separated by centrifugation (3000 rpm, 10 min). CK and LDH levels were measured in duplicate at each time point using a Japan Society of Clinical Chemistry Standardized Method [23], and the intra- and inter-assay CVs were 1.0 and 0.8 %, 0.9 and 0.8 %, respectively. Muscle soreness was evaluated using VAS, which is employed in various studies as a method to evaluate subjective muscle soreness [3, 5, 20, 21, 24]. A 100-mm scale from ‘no pain’ at 0 mm to ‘worst imaginable pain’ at 100 mm was set. Before measurement, the same examiner lightly pressed the subject’s biceps brachii at 7 cm above the elbow joint with a finger, and the subject marked his subjective muscle soreness on the scale. The measurement of the maximum isometric muscle strength of elbow flexion, the maximum static muscle strength of the non-dominant arm at an elbow joint angle of 90° was measured using Biodex System 3. Maximum contraction for 3 s was performed 3 times at 5-s resting intervals, and the highest value was adopted.
Statistical analysis
All data are presented as the mean ± confidence interval (CI) (except physical characteristics data: mean ± SD). Measurements data were converted to change scores, compared to pre value (Maximum isometric muscle strength values were converted to those per body weight). The physical characteristics and total load of eccentric contraction were analyzed using one-way ANOVA, and the other measurements were analyzed using two-way ANOVA with ingestion groups and time (group vs. time). When a main effect was detected, Tukey HSD multiple comparison was performed. The significance level was set at p less than 5 %. For the statistical analysis software, SPSS ver.20.0 Advanced Statistics (IBM, Co., USA) was used.