The impact of Omega-3 polyunsaturated fatty acid supplementation on exercise-induced muscle injury



Moral approval was obtained by the School of Liberal of Arts and Sciences Analysis Ethics Committee, College of Westminster (ETH1617–0182). All work herein conforms to the requirements set by the Declaration of Helsinki of 1975. Written knowledgeable consent was obtained from all members previous to their participation.

A complete of 23 wholesome, bodily lively males (self-reported: 4–5 occasions weekly structured train) aged 18–35 years of age had been recruited to take part on this experimental examine. Following withdrawals (n = 9; incapability to attend all visits, damage or sickness outdoors of trials or incapability to finish downhill protocol), 14 members (25.07 ± 4.05 years of age) accomplished the protocol and are included within the evaluation beneath.

Individuals had been required to chorus from any structured train for 48 h, and from alcohol and caffeine 24 h previous to baseline go to and EIMD protocol. They had been additionally requested to chorus from eccentric strenuous train through the 5 weeks of the examine in addition to within the following 72 h after the muscle-damaging train bout. Exclusion standards included age (outdoors 18–35 age vary), smoking, intercourse, taking any treatment (e.g. non-steroidal anti-inflammatory medication), and consuming fish oil dietary supplements < 6 months previous to commencing the examine and the presence of any identified immune, cardiovascular or metabolic illness. To additional affirm members had been free from higher respiratory tract infections, they accomplished an illness-specific questionnaire (WURSS-21) [24]. Moreover, members had been free from any ache or damage as decided by the Bodily Exercise Readiness Questionnaire (PAR-Q) pre-exercise participation screening. Individuals had been additionally excluded in the event that they repeatedly undertook downhill working or eccentric train (e.g. resistance train, squats) as a part of their regular coaching < 6 months previous to commencing the examine.

Experimental design

All members had been required to attend the human efficiency laboratory within the morning on 5 events. Throughout go to 1, in an in a single day fasted-state, members carried out baseline measurements which included anthropometric measurements, a urine pattern and a venous blood pattern. Perceived muscle soreness, maximal voluntary isometric contraction (MVIC) on the leg and anaerobic peak energy by way of Wingate take a look at had been decided as oblique markers of muscle injury, described totally beneath.

Following baseline measurements, members carried out a treadmill V̇O2max take a look at (HP Cosmos Mercury 4.0, Nussdorf-Traunstein, Germany) with expired gases analysed by an on-line breath-by breath system (Cortex Metalyser 3B, Biophysik, Leipzig, Germany). The Metalyser was calibrated in keeping with producer’s tips prior to every take a look at. Following completion of the V̇O2max take a look at, a 6-min working pace (Vtake a look at) at 65% V̇O2max verification was carried out on a downhill run at − 10% gradient, as we now have beforehand reported [25]. After baseline testing, members had been single-blind randomised to both N-3 (3 g/day of n-3 PUFA) or PLA (placebo) group by a computer-generated block randomization prematurely (

Two weeks earlier than starting testing, members crammed a well being questionnaire (WURSS-21) on every of the 14 days previous trial to make sure that they had been free from frequent chilly signs earlier than testing. In go to 2, members reported to the laboratory at 07:00 am having fasted in a single day and carried out the EIMD protocol (downhill working; 60 min at 65% V̇O2max with a − 10% gradient). All above measurements had been repeated prior to- and immediately-post the EIMD trial. Someday earlier than the go to members had been requested to eat water based mostly on their physique mass (5 mL/kg) [25] earlier than they reported to the laboratory to make sure sufficient hydration earlier than train. Similar comply with up assessments, besides urine pattern, had been repeated at visits 3-to-5 (24, 48 and 72 h post-EIMD), throughout which members had been in a non-fasted state. An summary of the examine design is introduced in Fig. 1.

Fig. 1

Schematic of experimental procedures: USG, urine particular gravity; BIA, bioelectrical impedance evaluation; VAS, visible analogue scale for delayed-onset muscle soreness; MVIC, maximal voluntary isometric contraction; V̇O2max, maximal oxygen consumption; N-3, omega-3 supplementation group; PLA, placebo group; EIMD, exercise-induced muscle injury. 2nd go to combines each pre and put up measurements, instantly prior and following EIMD stimulus, respectively. An extra Wingate take a look at was added on 2nd go to and all comply with up visits

Hydration standing

Urine pattern was collected at baseline and pre-EIMD to evaluate members’ hydration standing. Hydration was verified by checking that urine particular gravity (USG) (Atago MASTER-SUR/Na refractometer, Atago Co., Ltd. Tokyo, Japan) upon arrival was between 1.001 and 1.029 [26]. Urine color was additionally checked by utilizing the validated urine color chart (1–8 scale) [26].

Anthropometric measurements

Top (to nearest 0.1 cm) was measured utilizing a wall-mounted “Harpenden” stadiometer (Holtain Ltd., Crymych, Wales, UK) fitted with a excessive pace Veeder-Root counter (Veeder-Root, Elizabethtown, NC, USA) with members stood in naked toes, heels along with their shoulders and buttocks involved with the stadiometer. Physique weight (to nearest 0.1 kg), BMI and physique fats % (to nearest 0.1%) had been measured utilizing BIA (Tanita SC-330ST, Tokyo, Japan) with participant being fasted and with an empty bladder.


Omega-3 supplementation consisted of three gelatine-coated capsules per day (1 consumed within the morning, 1 at lunch and 1 within the night), every containing 1040 mg of n-3 PUFA (715 mg of EPA and 286 mg DHA) per capsule (Most Energy Pure Fish Oil, Nature’s Greatest, UK) for a complete of 3900 mg of fish oil each day, containing 3 g of n-3 PUFA (2145 mg of EPA and 858 mg DHA) per day for a interval of 4 weeks. While generally reported unwanted effects of n-3 supplementation, similar to disagreeable style, heartburn, gastrointestinal discomfort and headache are often delicate [27], the quantity of n-3 offered is consistent with the dietary suggestions as a part of a traditional food regimen and doesn’t trigger any hurt or unwanted effects. Day by day supplementation of as much as about 5 g/day of n-3 PUFA in a long-term consumption is taken into account protected by the European Meals Security Authority (EFSA) [28]. Longer length or excessive doses might have an effect on immune operate attributable to suppression of inflammatory response [29]. Excessive doses additionally may improve bleeding time by lowering platelet aggregation [29]. The placebo group obtained 3 × 600 mg capsules per day of collagen (Troo Healthcare, Colchester, UK), consumed in an identical sample. Individuals had been solely given 1 week of capsules at a time. Initially written reminders had been despatched every day to make sure supplementation practices had been maintained constant all through the day. Individuals’ compliance additionally verified by weekly written and oral reminders, counting remaining capsules on the finish of every week and issuing of future weeks capsules passed off. Additional, members had been requested to guess what group they had been on the conclusion of testing with 2 of seven in placebo and 5 of seven in N-3 group appropriately guessing the supplementation group.

Food regimen and exercise management

Individuals had been requested to take care of their ordinary food regimen and bodily exercise all through the examine. A 48-h meals diary (together with 1 day of the week and one weekend day) was offered to file all meals and drinks consumed previous to the supplementation interval beginning. Following 4 weeks of supplementation, members accomplished a second 48-h meals diary within the 2 days previous to the EIMD trial. Written and oral reminders had been additionally offered frequently to make sure food regimen and train practices had been maintained constant all through the examine.

Meals diaries had been analysed utilizing Nutritics® to quantify whole vitality consumption, macronutrients (carbohydrates, protein, fatty acids), n-3 and n-6 PUFA earlier than and after the supplementation interval. As well as, in the beginning of the supplementation interval all members had been supplied with a meals listing with meals low (< 250 mg per serving), average (~ 250 mg per serving) and excessive (> 500 mg per serving) in omega-3 fatty acids to stop growing their omega-3 consumption by way of food regimen. Lower-off factors had been utilized by USDA SR-21 (2008) [30].

EIMD protocol

Following a 3-min heat up, members ran for 60 min on the individualized predetermined Vtake a look at at − 10% gradient. Coronary heart fee (HR) and ranking of perceived exertion (RPE), Borg 6–20 scale [31] had been recorded all through the trial each 10 min. A 60-s pattern expired of gases had been collected at 20 and 40 min of trial and analysed by an on-line breath-by breath system for V̇O2 to make sure members had been working at 65% V̇O2max. Water was offered advert libitum each 15 min while working. Instantly after the muscle-damaging bout members sat and a blood pattern was collected (post-EIMD). Individuals then rated their perceived muscle soreness, and MVIC and Wingate take a look at had been carried out to evaluate members’ post-EIMD power and energy output, respectively.

Venous plasma

A complete of 12 mL of venous blood was collected at every time level in two 6 mL vacutainer tubes (Okay2 EDTA and lithium-heparin; BD, Oxford, UK). Haematocrit with capillary methodology utilizing a micro-hematocrit reader (Hawksley & Sons Ltd., Lancing, UK) and haemoglobin focus utilizing a photometer (Haemocue, Sheffield, UK) had been each analysed instantly on heparinized entire blood in triplicate. Subsequently, focus of plasma markers was adjusted for plasma quantity modifications with the tactic of Dill and Costill [32]. The remaining entire blood was spun (Hettich Common 320 R, Germany) at 5000 rpm for 10 min at 4 °C, with plasma aliquoted and frozen at − 80 °C.

Circulating CK exercise was measured utilizing a medical chemistry analyser (Werfen ILab Aries, Italy). CK exercise was decided utilizing kinetic spectrophotometry at 340 nm with a minimal detection restrict of three U/L, an undiluted linearity as much as 900 U/L. Coefficient of variation (CV) was inside run < 1.2%, whole < 2.5%. All samples and requirements had been analysed in duplicate.

Aliquots of plasma had been analysed for plasma IL-6 and TNF-α focus by enzyme-linked immunosorbent assay (ELISA) DuoSet kits and ancillary reagent packs (IL-6 DY206, TNF-α, DY210, consumables DY008, R&D Methods, USA) in duplicate, in keeping with producer’s instruction. Plates had been learn at 450 nm and blanked to 590 nm.

Perceived muscle soreness

Muscle soreness was self-rated by members on a 10-point-validated visible analogue scale (VAS) indicating on a line from 0 (no ache) to 10 (excessive ache) [33], throughout a wall squat with thighs parallel to the ground at 900 levels.

Maximal voluntary isometric contraction

MVIC was assessed on a dynamometer (Globus Kineo 7000, Italy). The chair was adjusted in order that the leg pad was positioned on the decrease a part of the tibialis anterior and the pivot was situated on the lateral epicondyle of the precise leg. Maximal pressure was measured at an angle of 60o leg extension. Peak pressure was decided by the common of 4 maximal isometric contractions lasting 3–5 s. The contraction time was recorded by an experimenter.

Peak energy

Individuals carried out a ten s Wingate take a look at on a cycle ergometer (Monark Ergomedic 894E, Sweden), fastened with an optical sensor (OptoSensor 2000™, Sport Medication Industries, USA) with the info obtained by the Monark Anaerobic Check Software program. Individuals cycled seated through the dash protocol, with a resistance equal to 7.5% of their physique weight. Individuals had been verbally inspired all through the take a look at.

Statistical evaluation

Regular distribution of all information was carried out by the Shapiro-Wilk Check. Following Levene’s take a look at of equality of variance, baseline traits, dietary and hydration information had been in contrast between teams utilizing a two-tailed impartial samples t-test. The examination of the impact of the n-3 supplementation on plasma CK exercise, IL-6, TNF-α and DOMS was carried out by non-parametric assessments, as these variables didn’t comply with regular distribution. Mann-Whitney U take a look at was carried out to look at variations between N-3 and PLA group at every time level. A Freidman take a look at was used to find out the primary impact of time within-group and put up hoc with Wilcoxon Signed Rank assessments (utilizing a Bonferroni adjusted alpha worth) was run the place a major time was recognized. MVIC and peak energy information met all assumptions required for normality and had been analysed utilizing a two-way combined between-within participant repeated measures evaluation of variance (ANOVA). Bonferroni-adjust pairwise comparisons put up hoc evaluation was used the place wanted to look at inside topic variations. Values had been expressed as imply ± SD for information from parametric assessments and as median and interquartile vary for information from non-parametric assessments. Statistical significance was accepted as p < 0.05. Impact measurement was calculated utilizing strategies proposed by Cohen [34], with impact sizes thought of small (0.2), medium (0.5) or massive (0.8). Statistical analyses had been carried out utilizing SPSS 25 software program (IBM SPSS, NY, USA). All figures had been generated in GraphPad Prism (Model 8, GraphPad).

Energy calculation

The pattern measurement was estimated from a pattern calculation (G*Energy 3.1) with an alpha stage of 0.05, an influence (1-β) of 0.80 and a medium impact measurement of 0.5 and suggests n = 12 in whole could be enough.