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Supplementation of eicosapentaenoic acid-rich fish oil attenuates muscle stiffness after eccentric contractions of human elbow flexors

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  • Supplementation of eicosapentaenoic acid-rich fish oil attenuates muscle stiffness after eccentric contractions of human elbow flexors


    A total of 16 healthy men were recruited for this study. The participants were not allergic to fish and had no resistance training. Thus, they were requested 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 an informed consent was obtained. The present study was performed in accordance with the Declaration of Helsinki and was approved by the ethics committee for human experiments of Hosei University (ID: 2017–002). Moreover, it has been registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR identifier: UMIN000028165).

    Study design

    The study used the double-blind, placebo-controlled, parallel-group trial design. Since this study design was similar to our previous studies [2, 5] in order to extend these previous studies, we conducted similar type of subjects and ECCs. The participants were randomly assigned to two groups using a table of random numbers to minimize the intergroup differences in terms of age, and body mass index (BMI). According to the previous studies [2, 5], the placebo (PL) group consumed daily placebo capsules for 8 weeks prior to an exercise experiment and for 5 days after the exercise, whereas the EPA group consumed EPA supplement capsules. The participants consumed the capsules for 62 days (including during exercise days). 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, the markers of muscle damage were assessed using the nondominant arm before exercise. Immediately after these baseline measurements, the participants performed ECCs using the same arm. All measurements were performed immediately before exercise, immediately after exercise, and 1, 2, and 5 days after exercise. In addition, the nutrition status of all participants was assessed 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). In addition, serum fatty acid levels were measured, including EPA, DHA, arachidonic acid (AA), and dihomo-gamma-linolenic acid (DGLA) levels.


    Based on previous studies [2, 3, 5] and considering the safety factor [14], the EPA group consumed eight 300-mg EPA-rich fish oil softgel capsules (Nippon Suisan Kaisha Ltd., Tokyo, Japan) per day, and the total consumption was 2400 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 2400 mg. The participants took the capsules within 30 min after each meal.

    Blood sample

    The participants fasted for 8 h before a trained doctor obtained blood samples from the forearm [5]. The blood samples were allowed to clot at room temperature (25 C) and were then centrifuged at 3000 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.


    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) measurement at 90 was converted to kg. The exercise consisted of 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 [2]. 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 ECC.

    MVC torque

    For the measurement of MVC torque, the participant performed three 3-s MVCs at 90, 110, 130 of elbow joint angle with a 15-s rest during contractions. The peak torque of each angle contractions 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 with a 16-bit analog-to-digital converter (Power-Lab 16SP; AD Instruments, Bella Vista, Australia). The sampling frequency was set at 10 kHz. The measurement was based on a previous study [15].

    ROM 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 [5, 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.

    Muscle soreness

    Muscle soreness in the elbow flexors was assessed using a 10-cm visual analog scale in which 0 indicated “no pain” and 10 “the worst pain imaginable” [5, 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.

    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 the arms relaxed by their side [2]. 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.

    Muscle echo intensity

    For the measurement of muscle echo intensity, the elbow joint angles of the participants were set at 70, 110, and 150. The B-mode ultrasound pictures of the upper arm were obtained using the biceps brachii via an ultrasound (Aixplorer version 4.2, Supersonic Imagine, France), 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 gray scale histogram (0, black; 100, white) for the region, as described in a previous study [2].

    Muscle stiffness

    Using the ultrasound shear wave elastography, muscle stiffness at 70, 110, and 150 elbow angle were measured, as previously described [6]. An ultrasonic scanner (Aixplorer version 4.2, Supersonic Imagine, France) in shear wave elastography mode with musculoskeletal preset was used. An electronic linear array probe (SL15–4, Supersonic Imagine) coated with water-soluble transmission gel was placed longitudinally on each muscle head. Muscle shear modulus (μ), a measure of normalized muscle stiffness, was calculated using the following equation: μ = ρVs2, where ρ is the muscle density (assumed to be 1000 kg/m3) and Vs is the velocity of shear wave propagation caused by focused ultrasound beam from the scanner. A 10-mm square map of the muscle shear modulus with a spatial resolution of 1 1 mm was obtained with each ultrasound image. A representative value of the shear modulus for each muscle head was then identified via spatial averaging over a 5-mm diameter circle [18].

    Statistical analyses

    All analyses were performed using the SPSS software version 22.0 (IBM Corp., Armonk, NY). Values were expressed as means standard deviation. MVC torque, ROM, muscle soreness, upper arm circumference, muscle echo intensity, and muscle stiffness of values on the time immediately after exercise, the days 1, 2, and 5 post-exercise were calculated based on relative changes from baseline. MVC, ROM, muscle soreness, upper arm circumference, muscle echo intensity, and muscle stiffness of the PL and EPA groups were compared using two-way repeated-measure analysis of variance. When a significant primary effect or interaction was found, Bonferroni’s correction was performed for post-hoc testing. The partial eta squared (η2) were calculated to demonstrate the effect size. A p-value of
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