Participants
Ethical approval was obtained by the College of Liberal of Arts and Sciences Research Ethics Committee, University of Westminster (ETH1617–0182). All work herein conforms to the standards set by the Declaration of Helsinki of 1975. Written informed consent was obtained from all participants prior to their participation.
A total of 23 healthy, physically active males (self-reported: 4–5 times weekly structured exercise) aged 18–35 years of age were recruited to participate in this experimental study. Following withdrawals (n = 9; inability to attend all visits, injury or illness outside of trials or inability to complete downhill protocol), 14 participants (25.07 ± 4.05 years of age) completed the protocol and are included in the analysis below.
Participants were required to refrain from any structured exercise for 48 h, and from alcohol and caffeine 24 h prior to baseline visit and EIMD protocol. They were also asked to refrain from eccentric strenuous exercise during the 5 weeks of the study as well as in the following 72 h after the muscle-damaging exercise bout. Exclusion criteria included age (outside 18–35 age range), smoking, sex, taking any medication (e.g. non-steroidal anti-inflammatory drugs), and consuming fish oil supplements < 6 months prior to commencing the study and the presence of any known immune, cardiovascular or metabolic disease. To further confirm participants were free from upper respiratory tract infections, they completed an illness-specific questionnaire (WURSS-21) [24]. Additionally, participants were free from any pain or injury as determined by the Physical Activity Readiness Questionnaire (PAR-Q) pre-exercise participation screening. Participants were also excluded if they regularly undertook downhill running or eccentric exercise (e.g. resistance exercise, squats) as part of their normal training < 6 months prior to commencing the study.
Experimental design
All participants were required to attend the human performance laboratory in the morning on 5 occasions. During visit 1, in an overnight fasted-state, participants performed baseline measurements which included anthropometric measurements, a urine sample and a venous blood sample. Perceived muscle soreness, maximal voluntary isometric contraction (MVIC) on the leg and anaerobic peak power via Wingate test were determined as indirect markers of muscle damage, described fully below.
Following baseline measurements, participants performed a treadmill V̇O2max test (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 according to manufacturer’s guidelines prior to each test. Following completion of the V̇O2max test, a 6-min running speed (Vtest) at 65% V̇O2max verification was performed on a downhill run at − 10% gradient, as we have previously reported [25]. After baseline testing, participants were single-blind randomised to either N-3 (3 g/day of n-3 PUFA) or PLA (placebo) group by a computer-generated block randomization in advance (http://www.randomization.com).
Two weeks before beginning testing, participants filled a health questionnaire (WURSS-21) on each of the 14 days preceding trial to ensure that they were free from common cold symptoms before testing. In visit 2, participants reported to the laboratory at 07:00 am having fasted overnight and performed the EIMD protocol (downhill running; 60 min at 65% V̇O2max with a − 10% gradient). All above measurements were repeated prior to- and immediately-post the EIMD trial. One day before the visit participants were asked to consume water based on their body mass (5 mL/kg) [25] before they reported to the laboratory to ensure adequate hydration before exercise. Identical follow up assessments, except urine sample, were repeated at visits 3-to-5 (24, 48 and 72 h post-EIMD), during which participants were in a non-fasted state. An overview of the study design is presented in Fig. 1.
Hydration status
Urine sample was collected at baseline and pre-EIMD to assess participants’ hydration status. Hydration was verified by checking that urine specific gravity (USG) (Atago MASTER-SUR/Na refractometer, Atago Co., Ltd. Tokyo, Japan) upon arrival was between 1.001 and 1.029 [26]. Urine colour was also checked by using the validated urine colour chart (1–8 scale) [26].
Anthropometric measurements
Height (to nearest 0.1 cm) was measured using a wall-mounted “Harpenden” stadiometer (Holtain Ltd., Crymych, Wales, UK) fitted with a high speed Veeder-Root counter (Veeder-Root, Elizabethtown, NC, USA) with participants stood in bare feet, heels together with their shoulders and buttocks in contact with the stadiometer. Body weight (to nearest 0.1 kg), BMI and body fat % (to nearest 0.1%) were measured using BIA (Tanita SC-330ST, Tokyo, Japan) with participant being fasted and with an empty bladder.
Supplementation
Omega-3 supplementation consisted of 3 gelatine-coated capsules per day (1 consumed in the morning, 1 at lunch and 1 in the evening), each containing 1040 mg of n-3 PUFA (715 mg of EPA and 286 mg DHA) per capsule (Maximum Strength Pure Fish Oil, Nature’s Best, UK) for a total of 3900 mg of fish oil daily, containing 3 g of n-3 PUFA (2145 mg of EPA and 858 mg DHA) per day for a period of 4 weeks. Whilst commonly reported side effects of n-3 supplementation, such as unpleasant taste, heartburn, gastrointestinal discomfort and headache are usually mild [27], the amount of n-3 provided is in line with the nutritional recommendations as part of a normal diet and does not cause any harm or side effects. Daily supplementation of up to about 5 g/day of n-3 PUFA in a long-term consumption is considered safe by the European Food Safety Authority (EFSA) [28]. Longer duration or high doses may affect immune function due to suppression of inflammatory response [29]. High doses also might increase bleeding time by reducing platelet aggregation [29]. The placebo group received 3 × 600 mg capsules per day of collagen (Troo Healthcare, Colchester, UK), consumed in a matching pattern. Participants were only given 1 week of capsules at a time. Initially written reminders were sent on a daily basis to ensure supplementation practices were maintained consistent throughout the day. Participants’ compliance also verified by weekly written and oral reminders, counting remaining capsules at the end of each week and issuing of future weeks capsules took place. Further, participants were asked to guess what group they were at the conclusion of testing with 2 of 7 in placebo and 5 of 7 in N-3 group correctly guessing the supplementation group.
Diet and activity control
Participants were requested to maintain their usual diet and physical activity throughout the study. A 48-h food diary (including 1 day of the week and one weekend day) was provided to record all foods and drinks consumed prior to the supplementation period starting. Following 4 weeks of supplementation, participants completed a second 48-h food diary in the 2 days prior to the EIMD trial. Written and oral reminders were also provided on a regular basis to ensure diet and exercise practices were maintained consistent throughout the study.
Food diaries were analysed using Nutritics® to quantify total energy intake, macronutrients (carbohydrates, protein, fatty acids), n-3 and n-6 PUFA before and after the supplementation period. In addition, at the start of the supplementation period all participants were provided with a food list with foods low (< 250 mg per serving), moderate (~ 250 mg per serving) and high (> 500 mg per serving) in omega-3 fatty acids to prevent increasing their omega-3 intake through diet. Cut-off points were used by USDA SR-21 (2008) [30].
EIMD protocol
Following a 3-min warm up, participants ran for 60 min at the individualized predetermined Vtest at − 10% gradient. Heart rate (HR) and rating of perceived exertion (RPE), Borg 6–20 scale [31] were recorded throughout the trial every 10 min. A 60-s sample expired of gases were collected at 20 and 40 min of trial and analysed by an on-line breath-by breath system for V̇O2 to ensure participants were running at 65% V̇O2max. Water was provided ad libitum every 15 min whilst running. Immediately after the muscle-damaging bout participants sat and a blood sample was collected (post-EIMD). Participants then rated their perceived muscle soreness, and MVIC and Wingate test were performed to assess participants’ post-EIMD strength and power output, respectively.
Venous plasma
A total of 12 mL of venous blood was collected at each time point in two 6 mL vacutainer tubes (K2 EDTA and lithium-heparin; BD, Oxford, UK). Haematocrit with capillary method using a micro-hematocrit reader (Hawksley & Sons Ltd., Lancing, UK) and haemoglobin concentration using a photometer (Haemocue, Sheffield, UK) were both analysed immediately on heparinized whole blood in triplicate. Subsequently, concentration of plasma markers was adjusted for plasma volume changes with the method of Dill and Costill [32]. The remaining whole blood was spun (Hettich Universal 320 R, Germany) at 5000 rpm for 10 min at 4 °C, with plasma aliquoted and frozen at − 80 °C.
Circulating CK activity was measured using a clinical chemistry analyser (Werfen ILab Aries, Italy). CK activity was determined using kinetic spectrophotometry at 340 nm with a minimum detection limit of 3 U/L, an undiluted linearity up to 900 U/L. Coefficient of variation (CV) was within run < 1.2%, total < 2.5%. All samples and standards were analysed in duplicate.
Aliquots of plasma were analysed for plasma IL-6 and TNF-α concentration by enzyme-linked immunosorbent assay (ELISA) DuoSet kits and ancillary reagent packs (IL-6 DY206, TNF-α, DY210, consumables DY008, R&D Systems, USA) in duplicate, according to manufacturer’s instruction. Plates were read at 450 nm and blanked to 590 nm.
Perceived muscle soreness
Muscle soreness was self-rated by participants on a 10-point-validated visual analogue scale (VAS) indicating on a line from 0 (no pain) to 10 (extreme pain) [33], during a wall squat with thighs parallel to the floor at 900 degrees.
Maximal voluntary isometric contraction
MVIC was assessed on a dynamometer (Globus Kineo 7000, Italy). The chair was adjusted so that the leg pad was placed on the lower part of the tibialis anterior and the pivot was located on the lateral epicondyle of the right leg. Maximal force was measured at an angle of 60o leg extension. Peak force was determined by the average of four maximal isometric contractions lasting 3–5 s. The contraction time was recorded by an experimenter.
Peak power
Participants performed a 10 s Wingate test on a cycle ergometer (Monark Ergomedic 894E, Sweden), fixed with an optical sensor (OptoSensor 2000™, Sport Medicine Industries, USA) with the data obtained by the Monark Anaerobic Test Software. Participants cycled seated during the sprint protocol, with a resistance equal to 7.5% of their body weight. Participants were verbally encouraged throughout the test.
Statistical analysis
Normal distribution of all data was performed by the Shapiro-Wilk Test. Following Levene’s test of equality of variance, baseline characteristics, dietary and hydration data were compared between groups using a two-tailed independent samples t-test. The examination of the effect of the n-3 supplementation on plasma CK activity, IL-6, TNF-α and DOMS was performed by non-parametric tests, as these variables did not follow normal distribution. Mann-Whitney U test was performed to examine differences between N-3 and PLA group at each time point. A Freidman test was used to determine the main effect of time within-group and post hoc with Wilcoxon Signed Rank tests (using a Bonferroni adjusted alpha value) was run where a significant time was identified. MVIC and peak power data met all assumptions required for normality and were analysed using a two-way mixed between-within participant repeated measures analysis of variance (ANOVA). Bonferroni-adjust pairwise comparisons post hoc analysis was used where needed to examine within subject differences. Values were expressed as mean ± SD for data from parametric tests and as median and interquartile range for data from non-parametric tests. Statistical significance was accepted as p < 0.05. Effect size was calculated using methods proposed by Cohen [34], with effect sizes considered small (0.2), medium (0.5) or large (0.8). Statistical analyses were performed using SPSS 25 software (IBM SPSS, NY, USA). All figures were generated in GraphPad Prism (Version 8, GraphPad).
Power calculation
The sample size was estimated from a sample calculation (G*Power 3.1) with an alpha level of 0.05, a power (1-β) of 0.80 and a medium effect size of 0.5 and suggests n = 12 in total would be sufficient.