A total of 22 healthy recreational untrained men were recruited for this study. The participants were not allergic to fish and had not participated in any regular resistance training experience for at least one year before this study. Further, participants were asked not to participate in other clinical trials and interventions, such as massage, stretching, strenuous exercise, excessive consumption of food or alcohol, and intake of supplementations or medications during the experimental period. All participants were provided with detailed explanations of the study protocol prior to participation, and informed consent was obtained from all participants. The present study was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee for human experiments of Teikyo Heisei University (ID: R01-040). Moreover, the study was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR identifier: UMIN000038003).
The study used the double-blind, placebo-controlled, parallel-group trial design. The participants were randomly assigned to two groups using a table of random numbers to minimize the intergroup differences in terms of age, body fat, and body mass index (BMI). The placebo (PL) and EPA and DHA group consumed daily placebo or fish oil capsules for 4 weeks prior to an exercise experiment and for 5 days after the exercise experiment. The sequence allocation concealment and blinding of participants and researchers were maintained throughout this period. Medication adherence was assessed using the daily record of the patients and via pill count at the end of the study. On the day of exercise testing, muscle damage markers were assessed using the nondominant arm before exercise. Immediately after these baseline measurements, the participants performed ECCs using the same arm. Maximum voluntary contraction (MVC) torque, ROM, DOMS, circumference, muscle echo intensity, and thickness were measured immediately before and after exercise and 1, 2, 3, and 5 days after exercise. Serum CK and IL6 were measured before exercise and 1, 2, 3, and 5 days after exercise. In addition, we measured serum fatty acid levels at before and after 4 weeks supplementation. Subjects were instructed to eat a light meal > 2 h before arriving at the laboratory. In addition, they were asked to refrain from any exercise for 24 h, before the study visit. In addition, we assessed the nutrition status of all participants prior to supplement consumption and after the experimental testing on food frequency using a questionnaire based on food groups (FFQg version 3.5, Kenpakusha, Tokyo, Japan). Furthermore, we measured serum fatty acid levels, including EPA, DHA, arachidonic acid (AA), and dihomo-gamma-linolenic acid (DGLA) levels.
The EPA and DHA group consumed eight 300-mg EPA-rich fish oil softgel capsules (Nippon Suisan Kaisha Ltd., Tokyo, Japan) per day, and the total consumption was 2,400 mg per day (600-mg EPA and 260-mg DHA). The PL group consumed eight 300-mg corn oil softgel capsules per day (without EPA and DHA), and the total consumption was 2,400 mg. The participants consumed the capsules within 30 min after the morning meal.
The participants fasted for 8 h before a trained doctor obtained blood samples from their forearms. The blood samples were allowed to clot at room temperature (25°C) and were then centrifuged at 3,000 rpm for 10 min at 4°C. The serum was extracted and stored at −20°C until analysis. The serum levels of DGLA, AA, EPA, and DHA were measured. In addition, we evaluated serum creatine kinase (CK) and interleukin-6 (IL-6) as muscle damage markers, as previously described 7.
For the ECCs, the participant sat on a preacher curl bench with his shoulder joint angle at 45° flexion. For the use of the dumbbell, the value of maximal voluntary contraction (MVC) torque measurement at 90° was converted to kilograms. The exercise comprised six sets of 10 maximal voluntary ECCs of the elbow flexors with a rest period of 90 s between each set, as described in our previous study 11. The dumbbell was handed to the participant at the elbow flexed position (90°), and the participant was instructed to lower it to a fully extended position (0°) at an approximately constant speed (30°/s) in time (3 s) with a metronome. The investigator then removed the dumbbell, and the participant returned his arm without the dumbbell to the start the position for the next ECCs.
Maximum voluntary contraction torque
For the measurement of MVC torque, each subject was seated with nondominant arm attached to a custom-designed ergometer and that performed isometric of the elbow flexors. The MVC torque was measured three 3-s MVCs at 90° and 110° of elbow joint angle with a 30-s rest during contractions. Subjects were verbally encouraged to give their maximal effort during the muscle strength tests. The greatest 1-s average of the three trials for each angle was used for subsequent analysis. The peak torque of each angle contraction was used as the MVC torque. The torque signal was amplified using a strain amplifier (LUR-A-100NSA1; Kyowa Electronic Instruments, Tokyo, Japan). The analog torque signal was converted to digital signals using a 16-bit analog-to-digital converter (Power-Lab 16SP; AD Instruments, Bella Vista, Australia). The sampling frequency was set at 10 kHz. The test–retest reliability of the MVC measures based on coefficient of variation (CV) was 2.8%.
Range of motion of the elbow joint
To examine the ROM of the elbow joint, two elbow joint angles (extended and flexed) were measured using a goniometer (Takase Medical, Tokyo, Japan). The extended joint angle was recorded while the participant attempted to fully extend the joint with the elbow held by his side and the hand in supination 7, 16, 17. The flexed joint angle was identified while the participant attempted to fully flex the joint from an equally fully extended position with the hand supinated. The ROM was calculated by subtracting the flexed joint angle from the extended joint angle. The test–retest reliability of the ROM measures based on CV was 2.2%.
Muscle soreness in the elbow flexors was assessed using a 10-cm visual analogue scale in which 0 indicated “no pain” and 10 indicated “unbearable imaginable” 7, 16, 17. The participant relaxed his arm in a natural position. The investigator then palpated the upper arm using a thumb, and the participant indicated his pain level using the visual analogue scale. All tests were conducted by the same investigator who had been trained to use the same pressure over time between participants. The test–retest reliability of the VAS measures based on CV was 1.9%.
Upper arm circumference
Upper arm circumference was assessed at 9 cm above the elbow joint using a tape measure while the participants were standing with their arms relaxed by their side 9. The measurement marks were maintained during the experimental period using a semipermanent ink marker, and a well-trained investigator obtained the measurements. The average value of the three measurements was used for further analysis. The test-retest reliability of the circumference measurements based on CV was 2.2%.
Muscle echo intensity and thickness
B-mode ultrasound pictures of the upper arm were obtained using the biceps brachii via an ultrasound (SONIMAGE HS1, Konika Minolta, Japan), and the probe was placed 9 cm from the elbow joint at the position marked for the measurement of the upper arm circumference. The same gains and contrast were used over the experimental period. The transverse images were transferred to a computer as bitmap (.bmp) files and analyzed using a computer. The average muscle echo intensity of the region of interest (20 × 20 mm) was calculated using the computer image analysis software that provided a grayscale histogram (0, black; 100, white) for the region, as described in a previous study (ImageJ, Maryland, USA) 9. Scanned images of each muscle were transferred to a personal computer and the thickness of biceps rachii was manually calculated via tracing using same software. The test-retest reliability of the muscle echo intensity and thickness measurements based on CV were 2.1% and 1.4%, respectively.
All analyses were performed using the SPSS software version 25.0 (IBM Corp., Armonk, NY). Values were expressed as means ± standard deviation. MVC torque, ROM, circumference, echo intensity, and thickness of values before exercise to 5 days post-exercise were calculated based on relative changes from the baseline. MVC, ROM, visual analogue scale (VAS), circumference, echo intensity, thickness, and blood data of the PL and EPA and DHA groups were compared using two-way repeated-measure analysis of variance (ANOVA). When a significant primary effect or interaction was found, Bonferroni’s correction was performed for post-hoc testing. A p-value of < 0.05 was considered statistically significant.