Krill oil is a rich source of the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which may alter immune function after exercise. The aim of the study was to determine the effects of krill oil supplementation on post exercise immune function and performance.
Nineteen males and 18 females (age: 25.8 ± 5.3 years; mean ± S.D.) were randomly assigned to 2 g/day of krill oil (n = 18) or placebo (n = 19) supplementation for 6 weeks. A maximal incremental exercise test and cycling time trial (time to complete set amount of work) were performed pre-supplementation with the time trial repeated post-supplementation. Blood samples collected pre- and post- supplementation at rest, and immediately, 1 and 3h post-exercise. Plasma IL-6 and thiobarbituric acid reactive substances (TBARS) concentrations and, erythrocyte fatty acid composition were measured. Natural killer (NK) cell cytotoxic activity and peripheral blood mononuclear cell (PBMC) IL-2, IL-4, IL-10, IL-17 and IFNγ production were also measured.
No effects of gender were noted for any variable. PBMC IL-2 and NK cell cytotoxic activity were greater (P < 0.05) 3h post exercise in the krill oil compared to the control group. Plasma IL-6 and TBARS, PBMC IL-4, IL-10, IL-17 and IFNγ production, along with performance and physiological measures during exercise, were not different between groups.
Citation: Da Boit M, Mastalurova I, Brazaite G, McGovern N, Thompson K, Gray SR (2015) The Effect of Krill Oil Supplementation on Exercise Performance and Markers of Immune Function. PLoS ONE 10(9): e0139174. https://doi.org/10.1371/journal.pone.0139174
Editor: Andrew Philp, University of Birmingham, UNITED KINGDOM
Received: June 17, 2015; Accepted: September 8, 2015; Published: September 25, 2015
Copyright: © 2015 Da Boit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: The study was approved by the University of Aberdeen College of Life Sciences and Medicine Ethics Review Board and data are ethically restricted for reasons related to patient privacy. Any data requests can be sent to the corresponding author, Dr. Stuart Gray (email@example.com), who will liaise with the ethics committee regarding data access. Any interested researcher can obtain a de-identified, minimal dataset pending ethical approval.
Funding: This work was supported with funding from Aker Biomarine Antarctic AS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: This study received financial support from Aker Biomarine AS. This did not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Fish oils are rich in the long chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to have positive effects in chronic inflammatory disease, such as cardiovascular disease  and diabetes mellitus . PUFAs are essential constituents of cell membranes of immune cells and are precursors of inflammatory mediators, such as prostaglandins and leukotrienes (for review see ). The primary PUFA found in cell membranes of leukocytes is the n-6 PUFA arachidonic acid (AA). However, increasing dietary consumption of EPA and DHA will alter the cell membrane composition and increase EPA and DHA levels with a concomitant reduction in the amount of AA . This alteration in fatty acid composition alters the substrate used for eicosanoid synthesis, resulting in a reduction in pro-inflammatory AA-derived mediators and an increase in the less potent EPA/DHA-derived alternative inflammatory mediators, such as the 3-, rather than the 2-, series prostaglandins . Fish oils are not the only source of EPA and DHA with Antarctic krill also being a rich source of n-3 PUFAs. The primary difference between fish and krill oil is that while fish oil contains the majority of n-3 PUFAs as triacylglycerol (TG) krill oil consists of more than 40% of n-3 PUFAs as phospholipids (PL) . Krill oil also contains the antioxidant astaxanthin. During supplementation studies, similar increases in plasma EPA and DHA can be seen when a third less PUFAs are given as krill compared to fish oil supplements . In another study when similar amounts of fish oil and krill oil, 600mg/day, are given the increases in plasma and erythrocyte EPA and DHA were found to be greater with krill compared to fish oil .
Fish oil supplements have been recommend for athletic populations due to evidence of their anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic and antiproliferative properties. It is thus suggested that the majority of athletes, especially those at leisure level, should consume 1–2 g/day of EPA and DHA to prevent muscle and joint inflammation and improve overall health. Over time this would not only improve overall health but may ultimately improve performance . In spite of these assertions the evidence to support the use of n-3 PUFAs specifically in exercising populations is limited and there is also concern that due to their high number of double bonds an increase in n-3 PUFA consumption may result in an increase in lipid peroxidation.
Whilst both fish and krill oil are rich n-3 PUFAs the majority of previous research in this area has utilised fish oil supplementation. One area where n-3 PUFA supplements may be useful is in the post-exercise recovery period where there are a variety of perturbations in the immune system . However there are a few studies which have investigated the effects of fish oil supplementation on various aspects of immune function after exercise. Toft and colleagues found that fish oil supplementation had no effect on plasma cytokine levels after exercise . One study found higher ex vivo activated peripheral blood mononuclear cell proliferation (PBMC) . In recent work we demonstrated that fish oil supplementation results in an increase in PBMC IL-2 production and natural killer (NK) cell cytotoxic activity in the recovery period after exercise in healthy young males . Whether this effect will be seen in females remains to be established. It has also been suggested that, via different mechanisms, n-3 PUFA supplementation may improve exercise performance. Indeed n-3 PUFA supplementation has been found to reduce HR and during exercise [14–17], although our findings do not support this . Furthermore these cardiometabolic changes do not translate into improvements in exercise performance [14, 16, 18], although one study did find an increase in after fish oil supplementation . The effects of krill oil on cardiometabolic variable and exercise performance remain to be established.
The aim of the present study was to investigate the effects of six weeks of supplementation with krill oil on markers of immune function (NK cell cytotoxicity and PBMC Th1/Th2 cytokine production) following an acute bout of endurance exercise in both males and females. A secondary aim was to determine whether krill oil supplementation altered exercise performance during a simulated cycling time trial.
Nineteen males and 18 females (aged 25.8 ± 5.3 years, height 175 ± 11.9 cm, weight 72.5 ± 14.9 kg; mean ± S.D.) volunteered to participate in the study. The study was approved by the University of Aberdeen College of Life Sciences and Medicine Ethics Review Board and participants were made aware of the aims, risks and potential discomfort associated with the study before providing written informed consent.
Participants were randomly assigned to either a placebo (n = 19) or krill oil (n = 18) group. Capsules were closely matched for both colour and shape, and both participants and investigators were blind to the supplementation group. After the preliminary maximal exercise test, participants in the placebo group consumed 2g (4*500mg) of placebo oil daily, while those in the krill oil group consumed 2g (4*500mg) of krill oil daily for a six week period. All capsules were provided by Aker Biomarine Antarctic AS (Oslo, Norway). The placebo oil was produced to reflect the fatty acid composition of the average European diet . Each 500mg krill oil capsule contained 60mg EPA, 30mg DHA and 61μg astaxanthin. To put this into a dietary context a 140g portion of oily fish would typically provide approximately 2.8 g EPA plus DHA, although this is known to vary dramatically between wild and farmed fish due to recent changes in feeds .
Maximal Exercise Test
For the 24h period prior to each visit, participants were instructed to refrain from the consumption of alcohol, caffeine and strenuous exercise. For the same period before the maximal exercise test, participants were asked to record their dietary intake and replicate this prior to the post-supplementation trial. All tests began at the same time of day, between 7 and 10am. A fasting blood sample was collected before performing an incremental maximal exercise test on a cycle ergometer (Lode, Netherlands). Participants cycled at self-selected cadence (70–90 rpm) with workload increasing by 30 Watts every minute for males and 20 Watts every minute for females until volitional exhaustion. Gas exchange (Medical Graphics, UK) and heart rate (Polar, Finland) were monitored throughout the test. was taken as the highest measured over a 30 second period and a workload estimated to elicit 70% calculated to be used in the exercise trials.
Baseline Exercise Trial
The exercise trial was a simulated time trial with the ergometer set in the linear mode. Approximately 7 days prior to this trial a familiarisation trial was carried out where no physiological measures were made but the participant completed the time trial as below. After a 5 min warm up at 75 Watts subjects performed a set amount of work in as quick a time as possible. The amount of work was based on the results of the maximal exercise test as:
This factor was chosen so that 70% of Wmax would be achieved at around 80rpm which is the preferred pedal rate, in our experience, in this population. During the time trial participants did not receive any information on workload, pedal rate, time or heart rate with only the amount of work completed and the amount remaining passed on to participants. Heart rate and gas exchange (3 min recording) were recorded at approximately 25, 50 and 75% of the percentage of work performed. Water was available ad libitum. This trial was the same as the post-supplementation trial, except no blood samples were collected during this trial.
After the 6-week supplementation period, fasted blood samples were taken. Participants then performed the time trial as in the baseline exercise trial. Immediately on cessation of exercise a post-exercise blood sample was drawn. During a 3h recovery period, participants remained fasted, and further blood samples were taken 1h and 3h post-exercise. Throughout the post-supplementation trial heart rate and gas exchange were recorded in the same way as during the baseline exercise trial. Participants were permitted to consume water ad libitum.
Blood samples were drawn from an antecubital vein using a butterfly needle (21G). Blood was collected in vacutainers coated with Lithium-Heparin (BD, Oxford, UK), for peripheral blood mononuclear cell (PBMC) isolation, and K+EDTA (BD, Oxford, UK) for separation of plasma. The K+EDTA vacutainer was centrifuged at 800g for 10 minutes at 4°C and plasma stored at -80°C until analysis.
Erythrocyte Fatty Acid Composition Analysis
Fatty acid analysis was performed in EDTA blood samples sent to a commercial laboratory (Omegametrix) and analysed as previously described . Samples were collected in 4 ml EDTA vacutainers, mixed and immediately shipped to Germany for analysis. Briefly, fatty acid methyl esters were generated from erythrocytes by acid transesterification and analysed by gas chromatography using a GC2010 Gas Chromatograph (Shimadzu, Duisburg, Germany) equipped with a SP2560, 100-m column (Supelco, Bellefonte, PA) using hydrogen as the carrier gas. Fatty acids were identified by comparison with a standard mixture of fatty acids characteristic of erythrocytes. A total of 26 fatty acids were identified and quantified. Results are given as percentage of total identified fatty acids after response factor correction. The coefficient of variation for EPA plus DHA and for most other fatty acids was ~4%. Analyses were quality-controlled according to DIN ISO 15189.
Measurements were performed using commercially available high sensitivity ELISA kits (R&D Systems, UK) according to the manufacturer’s instructions.
Plasma thiobarbituric acid reactive substances (TBARS)
Measurements were performed using commercially available parameter assay kits (R&D Systems, UK) according to the manufacturer’s instructions. The principle of this assay is that malondialdehyde (MDA), from lipid peroxides, in the presence of heat and acid, reacts with 2-thiobarbituric acid to produce a coloured end product which can be measured in a spectrophotometric plate reader. For this reason TBARS results are expressed as MDA concentration.
PBMC Isolation and Incubation
These procedures were carried out using the same methods as our previous work  where the protocol was sufficient to show a robust stimulation of cytokine production. Twenty ml whole blood was mixed with 10ml PBS and layered onto 20ml Histopaque (Sigma-Aldrich, St Louis, USA) before being centrifuged at 400g for 30 minutes at room temperature. The PBMC layer was collected and mixed with 40ml PBS and centrifuged at 250g for 10 minutes. The supernatant was then removed and the cell pellet reconstituted with 20ml PBS. This washing phase was then repeated. The final pellet was reconstituted with 1ml RPMI-1640 medium and cell numbers determined. 1.5x106 cells, in duplicate, were then incubated at 37°C and 5% CO2 for 24h supplemented with 2mM glutamine, antibiotics, 2.5% autologous plasma and 10 mg/L concanavalin A (Con A). At the end of incubation, each well was centrifuged at 250g for 10 minutes, the supernatant removed and frozen at -80°C for analysis of PBMC cytokine production.
PBMC Cytokine Production
All measurements were performed using commercially available multiplex assay kits (R&D Systems, UK) according to the manufacturer’s instructions (Luminex, USA). The cytokines IL-2, IL-4, IL-10, IL-17 and IFN-γ were measured in culture supernatants.
Natural Killer Cell Cytotoxic Activity
Natural killer cell cytotoxic activity was measured in PBMCs, isolated as above but prior to treatment/incubation, using NKTEST kits (Opregen Pharma, Heidelberg, Germany) according to the manufacturer’s instructions. While this kit will measure primarily NK cell cytotoxic activity other cell types with NK cell-like activity, ie γδ T cells, will make a minor contribution. Briefly, PBMCs were mixed with pre-stained K562 target cells in ratios of 50:1 and 25:1. Tubes were centrifuged for 3 min at 120g and then incubated for 2h in a CO2 incubator at 37°C. 50μl of DNA staining solution was added to the cells, which were incubated in the dark on ice for 5 min before analysis by flow cytometry. 2,500 cells were analysed for each sample, allowing us to determine the effects of exercise and krill oil. To discriminate target and effector cells a gate was set in the green fluorescence histogram. The percentage of dead cells was then determined using the red fluorescence histogram and the percentage specific cytotoxicity determined by subtracting the percentage dead cells in a tube containing K562 cells alone from the percentage of dead target cells in the samples.
Data analysis was carried out using SPSS software. Baseline characteristic data were compared between groups using independent t-tests. Initial analyses by sex found no effects and therefore are not presented here. Data were then analysed using a two way (time and group) repeated measures ANOVA with Bonferroni corrected post-hoc t-tests performed where appropriate. Statistical significance was accepted at P <0.05. All data are expressed as mean ± SD.
Subject baseline characteristics are summarised in Table 1. Baseline physical characteristics were similar between the groups.
Erythrocyte fatty acid composition
There were no differences in erythrocyte fatty acid composition between groups at baseline. In the control group there were no changes in erythrocyte fatty acid composition over the supplementation period. However in the krill oil group there were significant increases (P<0.05) in levels of C20:5n3, C22:6n3 and the Omega-3 index, with decreases (P<0.05) in levels of C20:4n6 and C22:4n6 (Table 2).
Time trial variables
The ANOVA revealed no time (F(3,105) = 3.67, P = 0.07), group (F(1,35) = 0.005, P = 0.94) or interaction (F(3,105) = 2.42, P = 0.12) effect for time to completion during the time trial. In the control group the time to complete the baseline time trial was 80.5 ± 11.2 min and 84.7 ± 17.1 min after the supplementation period. In the krill oil group baseline time to complete was 83.9 ± 14.7 min and 85.4 ± 19.8 min after the supplementation period. Similarly no time (HR: F(3,105) = 3.01, P = 0.09; , P = 0.07), group (HR: F(1,35) = 0.001, P = 0.97. , P = 0.50), or interaction (HR: F(3,105) = 1.22, P = 0.28. , P = 0.78) effects were noted for HR or during the time trial (Fig 1).
Plasma IL-6 and TBARS
The ANOVA for plasma IL-6 showed a time (F(3,105) = 7.6, P<0.05) effect with no group (F(1,35) = 0.45, P = 0.50) or interaction (F(3,105) = 0.40, P = 0.75) effects. Plasma IL-6 was higher (P<0.05) immediately, 1h and 3h post-exercise, compared to baseline, in both groups. There were no time (F(3,105) = 2.13, P = 0.10), group (F(1,35) = 0.15, P = 0.71) or interaction (F(3,105) = 0.12, P = 0.95) effects for plasma TBARS (Fig 2).
PBMC Cytokine Production
PBMC cytokine data is shown in Fig 3. The ANOVA revealed an interaction (F(3,105) = 3.17, P = 0.02) and time effect (F(3,105) = 6.43, P<0.05) for PBMC IL-2 production. Post-hoc analysis revealed that PBMC IL-2 was lower (P<0.05) than baseline, at 1h post-exercise in the control group and higher (P<0.05) than baseline, at 3h post-exercise in the krill oil group. Furthermore 3h post-exercise PBMC IL-2 was higher (P<0.05) in the krill oil compared to the control group. No group effect (F(1,35) = 1.11, P = 0.30) was observed for PBMC IL-2. Analysis showed an effect of time for PBMC IFN-γ (F(3,105) = 18.54, P<0.05) with post-hoc analysis revealing that PBMC IFN-γ was higher (P<0.05) immediately and after 3h, and lower at 1h, post-exercise compared with baseline in both groups. No group (F(1,35) = 0.02, P = 0.89) or interaction (F(3,105) = 0.94, P = 0.42) effect was observed for PBMC IFN-γ. The ANOVA revealed a time effect for PBMC IL-4 (F(3,105) = 3.27, P<0.05) with post hoc analysis showing that PBMC IL-4 was greater (P<0.05) at 1h and 3h post-exercise, compared with baseline, in the two groups. The analysis of PBMC IL-4 showed no interaction (F(3,105) = 0.26, P = 0.85) or group effect (F(1,35) = 0.04, P = 0.84). Analysis of PBMC IL-10 found an effect of time (F(3,105) = 5.763, P<0.05) with post-hoc analysis revealing that PBMC IL-10 was higher (P<0.05) 3h post-exercise compared with baseline in both groups. No group (F(1,35) = 0.24, P = 0.63) or interaction (F(3,105) = 0.59, P = 0.62) effect was observed for PBMC IL-10. The ANOVA revealed a time effect for PBMC IL-17 (F(3,105) = 12.86, P<0.05) with post-hoc analysis revealing that PBMC IL-17 was higher (P<0.05) at every time point compared with baseline, in both groups. No group (F(1,35) = 0.01, P = 0.91) or interaction (F(3,105) = 0.32, P = 0.81) effect was observed for PBMC IL-17.
NK cell function
Analysis of natural killer cell cytotoxic activity revealed a significant interaction effect at both 50:1 (F(3,105) = 8.18, P<0.05) and 25:1 (F(3,105) = 4.09, P<0.05) effector:target cell ratios. Post hoc analysis revealed that at 3h post-exercise NK cell cytotoxic activity was greater in the krill oil group, compared to the control group, for both effector:target ratios of 50:1 and 25:1 (P<0.05 Fig 4). In addition, time effects in NK cell cytotoxic activity at the 50:1(F(3,105) = 12.91, P<0.05) and 25:1 (F(3,105) = 13.77, P<0.05) ratios were observed. Post-hoc analysis found that NK cell cytotoxic activity was greater (P<0.05) at 1h post-exercise, compared to baseline, in the two groups and higher at 3h post-exercise, compared to baseline, in the krill oil group only. There was no effect of group for NK cell cytotoxic activity at either ratio (50:1−F(1,35) = 3.53, P = 0.07. 25:1−F(1,35) = 1.51, P = 0.22).
The current study has demonstrated that supplementation of the diet with 2 g per day of krill oil for 6 weeks increases the production of IL-2 in stimulated PBMCs and the cytotoxic activity of NK cells in the recovery period after exercise. These effects were seen in both males and females. There was no effect of krill oil supplementation on plasma levels of IL-6 and TBARS, or on PBMC production of IL-4, IL-10, IL-17 and IFNγ in response to exercise. Similarly, krill oil supplementation did not alter time trial performance or the oxygen cost and HR response to exercise.
The current study has demonstrated that 6 weeks of supplementation with krill oil results in a 75% increase in erythrocyte EPA, a 21% increase in DHA and a 27% increase in the Omega-3 Index. Alongside these increases in n-3 PUFA there were concomitant decreases of 7% and 17% in levels of arachidonic and docosatetraenoic acid, respectively. Therefore, our study is in agreement with previous work  which has demonstrated that krill oil supplementation at a level of around 1/3rd lower than previous work with fish oil can result in a similar magnitude of a rise in EPA and DHA. It is worth pointing out here that the current study made no direct comparison to a group of participants supplemented with fish oil. However, due to the lack of studies in this area with krill oil supplementation we have compared our studies to those where fish oil supplementation was given. That is not to say that fish and krill oil are the exact same, as highlighted in the introduction.
There were no effects of krill oil supplementation on any measures of immune function and lipid peroxidation at rest in the current study, which is in agreement with previous work with fish oil supplementation in healthy populations [4, 13, 23]. Others have shown that supplementation with increasing quantities of EPA tends to result in an increase in NK cell cytotoxic activity, although PBMC IL-2 production was not altered, in healthy young males . However, there are several studies in disagreement with this. In healthy older (>55y) participants it has been shown that 1.2 g/day EPA supplementation for 12 weeks can reduce NK cell cytotoxic activity . Similar findings were observed in young healthy men who were supplemented with 6g/day DHA for 90 days . Furthermore, in vitro studies of human NK cells and in young mice have found that EPA and DHA treatment reduces NK cell cytotoxic activity [27–29].
The current study has demonstrated that NK cell cytotoxic activity and PBMC IL-2, but not other cytokines, are increased after krill oil supplementation. What remains to be established are the mechanisms through which n-3 PUFA supplementation results in these alterations. It is known that PGE-2 can reduce the cytotoxic activity of human NK cells  and it is likely that the increase in EPA and DHA incorporation into immune cell membranes as a result of krill and fish oil supplementation will result in a concomitant decrease in membrane AA levels and thus altered eicosanoid synthesis . Primarily this would reduce PGE-2 synthesis and relieve its suppressive effects on NK cells, although why we do not then see a universal increase in NK cell activity with fish oil supplementation, in the above studies, is not clear. What is clear, from our work, is that both krill and fish oil supplementation results in an increase in PBMC IL-2 production and NK cell cytotoxic activity and as exercise increases PGE-2 production this may explain why we only see these positive effects with exercise. Indeed when indomethacin, which inhibits PGE-2 production, is administered during exercise a greater NK cell cytotoxic activity is observed . The current study has also found that there was no effect of krill oil on plasma IL-6 levels, which is in agreement with previous fish oil studies in this area [11, 13, 33].
There has been one study investigating the effects of krill oil supplementation on markers of oxidative stress and inflammation which, contrary to the current findings, demonstrated that in the recovery period after a bout of exercise, TBARS levels were lower in the krill oil group, compared to placebo . No differences in tumor-necrosis factor-alpha (TNF-α) or the activity of superoxide dismutase, glutathione peroxidase and creatine kinase were observed between groups. Previous work which has investigated the effects of fish oil supplementation on markers of oxidative stress is equivocal. Work by Bloomer and colleagues found that fish oil supplementation had no effect on plasma TBARS after 1h of treadmill exercise , which is in agreement with the current findings. However, it has also been shown that fish oil supplementation increase plasma F2-isoprostanes following three days of 3h cycling sessions , but plasma TBARS were found to be reduced after eccentric knee extensor exercise . It would appear therefore that EPA and DHA have no effect on markers of oxidative stress after acute submaximal exercise, but can reduce and increase such markers after acute eccentric and short-term intensified training, respectively.
The current data found that krill oil did not affect time trial performance, nor were heart rate or oxygen consumption affected. The fact that performance was not affected is in broad agreement with data published with fish oil where several studies have found no performance benefit, although cardiometabolic changes (ie reduced HR and ) were observed [14–17]. It is not clear why in the current study, and in our previous fish oil work, we have found no effect of n-3 PUFA supplementation on HR and . The human studies where these effects have been observed have in general been at either lower (i.e. 55% peak workload—compared to ~70% in the current study) or higher intensities (~10 min time trial to exhaustion) but it is not obvious why this would alter any effect of fish oil on these parameters. To clarify this it would be important to examine the effects of n-3PUFAs on HR and at varying exercise intensities within the same population. Together our data and that of others indicates that supplementation with n-3 PUFAs, either via fish or krill oil, does not alter exercise performance in young healthy volunteers. This lack of change in exercise performance may be due to the dose of krill, in the current study, and fish oil in previous studies, given not being sufficient to alter the fatty acid composition of muscle itself. Whilst we based our dose of krill oil on previous work comparing krill and fish oil  we have no data to show that this alter the muscle fatty acid composition. As measures of immune function were the primary outcome of the current study we chose not to collect muscle samples for such analysis but subsequent studies with a focus on exercise performance may wish to consider this. Furthermore it may also be the case that fish oil may still be of benefit for exercise performance in different populations, ie older people, or for different forms of exercise, ie strength training [37, 38]. On the other hand it may be, as the evidence suggests, that increasing n-3 PUFA consumption simply does not alter endurance exercise performance.
On top of the effects of krill oil supplementation on markers of immune function the current study also provides important information regarding the effects of exercise alone. Prior to discussion of these results, however, it is worth noting that we did not have a control group with no-exercise and so it is possible that these changes are circadian in origin. Our work would indicatively support recent findings that exercise results in an increase in the production of the T helper cell Th17 cytokine IL-17 , which has recently been identified in having an important role in the regulation of inflammation with precise roles still being identified . The precise role of this increase in PBMC IL-17 during exercise is yet to be established and further work is needed in this area. IL-17 is known to drive the production of IL-6 and PGE-2 in many cell types, such as epithelial, endothelial, and fibroblastic cells  and can also stimulate T cell proliferation and IL-2 production , indicating several areas where IL-17 may have important roles during exercise. Furthermore determining the cell type that is producing IL-17 and whether we are observing and increase in individual cell IL-17 production or just a greater number of cells, e.g. Th17 cells, producing IL-17. We have also extended the pioneering work of Ostrowski and colleagues, who found increases in plasma IL-10 after exercise , by showing that exercise increases PBMC IL-10 production. We have further added to basic exercise immunology research by showing that these changes are seen in both males and females. In fact the majority of previous research in exercise immunology has been in male volunteers and so the current study is the first to find that the effects of such exercise on selective markers of immune function are independent of gender.
In conclusion, the current study is the first study to investigate the effects of 6-weeks of krill oil supplementation on exercise performance, markers of immune function and lipid peroxidation, and has shown that krill oil can increase PBMC IL-2 production and NK cell cytotoxic activity 3h post-exercise in both healthy young males and females without modifying performance. It is worth noting at this point that ideally we would have taking blood samples not only during the final time trial but also during the initial time trial, allowing a more direct comparison of responses in immune measures after the supplementation period in the same individuals, this is a limitation of the current study. It also remains to be established whether these alterations in immune function can ultimately reduce the burden of upper respiratory tract infections which are elevated in those with high training loads . In that vein, our recent work has found that a supplement containing EPA, DHA, protein and vitamin D can reduce URTI symptoms, while not URTI numbers or duration, in active young males and females (3), although whether these effects are specifically due to EPA and DHA is not clear.
We thank the technical support of the Institute of Medical Sciences Musculoskeletal Programme and the Iain Fraser Cytometry Centre.
Conceived and designed the experiments: SG MDB KT. Performed the experiments: SG MDB IM NM GB KT. Analyzed the data: SG MDB IM NM GB KT. Contributed reagents/materials/analysis tools: SG KT. Wrote the paper: SG MDB IM NM GB KT.
- 1. Lemaitre RN, King IB, Mozaffarian D, Kuller LH, Tracy RP, Siscovick DS. n-3 polyunsaturated fatty acids, fatal ischemic heart disease, and nonfatal myocardial infarction in older adults: The Cardiovascular Health Study. American Journal of Clinical Nutrition. 2003; 77:319–25. pmid:12540389
- 2. Nettleton JA, Katz R. n-3 long-chain polyunsaturated fatty acids in type 2 diabetes: A review. Journal of the American Dietetic Association. 2005; 105:428–40. pmid:15746832
- 3. Calder PC. Polyunsaturated fatty acids and inflammatory processes: new twists in an old tale. Biochimie. 2009; 91:791–795 pmid:19455748
- 4. Yaqoob P, Pala HS, Cortina-Borja M, Newsholme EA, Calder PC. Encapsulated fish oil enriched in alpha-tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions. European journal of clinical investigation. 2000; 30:260–74. pmid:10692004
- 5. Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients. 2010; 2:355–74. pmid:22254027
- 6. Tou JC, Jaczynski J, Chen Y-C. Krill for Human Consumption: Nutritional Value and Potential Health Benefits. Nutrition reviews. 2007; 65:63–77. pmid:17345959
- 7. Ulven SM, Kirkhus B, Lamglait A, Basu S, Elind E, Haider T, et al. Metabolic effects of krill oil are essentially similar to those of fish oil but at lower dose of EPA and DHA, in healthy volunteers. Lipids. 2011; 46:37–46. pmid:21042875
- 8. Ramprasath V, Eyal I, Zchut S, Jones P. Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil. Lipids in Health and Disease. 2013; 12:178. pmid:24304605
- 9. Simopoulos AP. Omega-3 fatty acids and athletics. Curr Sports Med Rep. 2007; 6:230–6. pmid:17617998
- 10. Walsh NP, Gleeson M, Shephard RJ, Woods JA, Bishop NC, Fleshner M, et al. Position statement. Part one: Immune function and exercise. Exercise immunology review. 2011; 17:6–63. pmid:21446352
- 11. Toft AD, Thorn M, Ostrowski K, Asp S, Moller K, Iversen S, et al. N-3 polyunsaturated fatty acids do not affect cytokine response to strenuous exercise. Journal of applied physiology. 2000; 89:2401–6. pmid:11090595
- 12. Andrade PMM, Ribeiro BG, Bozza MT, Costa Rosa LF, do Carmo MGT. Effects of the fish-oil supplementation on the immune and inflammatory responses in elite swimmers. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2007; 77:139–45.
- 13. Gray P, Gabriel B, Thies F, Gray SR. Fish oil supplementation augments post-exercise immune function in young males. Brain, behavior, and immunity. 2012; 26:1265–72. pmid:22902522
- 14. Peoples GE, McLennan PL, Howe PR, Groeller H. Fish oil reduces heart rate and oxygen consumption during exercise. Journal of cardiovascular pharmacology. 2008; 52:540–7. pmid:19034030
- 15. Peoples GE, McLennan PL. Dietary fish oil reduces skeletal muscle oxygen consumption, provides fatigue resistance and improves contractile recovery in the rat in vivo hindlimb. The British journal of nutrition. 2010; 104:1771–9. pmid:20691135
- 16. Buckley JD, Burgess S, Murphy KJ, Howe PRC. DHA-rich fish oil lowers heart rate during submaximal exercise in elite Australian Rules footballers. Journal of Science and Medicine in Sport. 2009; 12:503–7. pmid:18555744
- 17. Kawabata F, Neya M, Hamazaki K, Watanabe Y, Kobayashi S, Tsuji T. Supplementation with eicosapentaenoic acid-rich fish oil improves exercise economy and reduces perceived exertion during submaximal steady-state exercise in normal healthy untrained men. Bioscience, biotechnology, and biochemistry. 2014; 78:2081–8. pmid:25144572
- 18. Oostenbrug GS, Mensink RP, Hardeman MR, De Vries T, Brouns F, Hornstra G. Exercise performance, red blood cell deformability, and lipid peroxidation: effects of fish oil and vitamin E. Journal of applied physiology. 1997; 83:746–52. pmid:9292459
- 19. Zebrowska A, Mizia-Stec K, Mizia M, Gasior Z, Poprzecki S. Omega-3 fatty acids supplementation improves endothelial function and maximal oxygen uptake in endurance-trained athletes. Eur J Sport Sci. 2015; 15:305–14. pmid:25176010
- 20. Bull NL, Day MJ, Burt R, Buss DH. Individual fatty acids in the British household food supply. Human nutritionApplied nutrition. 1983; 37:373–7.
- 21. (COT) SACoNSaCoT. Advice on fish consumption: benefits & risks. London, United Kingdom: Her Majesty's Stationery Office2004. https://doi.org/10.1016/j.envres.2014.10.029 pmid:25667172
- 22. von Schacky C, Kemper M, Haslbauer R, Halle M. Low Omega-3 Index in 106 german elite winter endurance athletes: a pilot study. International Journal of Sport Nutrition and Exercise Metabolism. 2014; 24:559–64. pmid:25203220
- 23. Gray P, Chappell A, Jenkinson AM, Thies F, Gray SR. Fish Oil Supplementation Reduces Markers of Oxidative Stress But Not Muscle Soreness After Eccentric Exercise. International Journal of Sport Nutrition and Exercise Metabolism. 2014.24:206–214 pmid:24225668
- 24. Miles EA, Banerjee T, Wells SJ, Calder PC. Limited effect of eicosapentaenoic acid on T-lymphocyte and natural killer cell numbers and functions in healthy young males. Nutrition (Burbank, Los Angeles County, Calif). 2006; 22:512–9.
- 25. Thies F, Nebe-von-Caron G, Powell JR, Yaqoob P, Newsholme EA, Calder PC. Dietary supplementation with eicosapentaenoic acid, but not with other long-chain n-3 or n-6 polyunsaturated fatty acids, decreases natural killer cell activity in healthy subjects aged >55 y. American Journal of Clinical Nutrition. 2001; 73:539–48. pmid:11237929
- 26. Kelley D, Taylor P, Nelson G, Schmidt P, Ferretti A, Erickson K, et al. Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men. Lipids. 1999; 34:317–24. pmid:10443964
- 27. Meydani SN, Yogeeswaran G, Liu S, Baskar S, Meydani M. Fish Oil and Tocopherol-Induced Changes in Natural Killer Cell-Mediated Cytotoxicity and PGE2 Synthesis in Young and Old Mice. The Journal of nutrition. 1988; 118:1245–52. pmid:3183771
- 28. Purasiri P, McKechnie A, Heys SD, Eremin O. Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids. Immunology. 1997; 92:166–72. pmid:9415022
- 29. Yamashita N, Yokoyama A, Hamazaki T, Yano S. Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid. Biochemical and biophysical research communications. 1986; 138:1058–67. pmid:3019330
- 30. Hall TJ, Chen SH, Brostoff J, Lydyard PM. Modulation of human natural killer cell activity by pharmacological mediators. Clinical and experimental immunology. 1983; 54:493–500. pmid:6317242
- 31. Calder PC, Yaqoob P, Thies F, Wallace FA, Miles EA. Fatty acids and lymphocyte functions. The British journal of nutrition. 2002; 87 Suppl 1:S31–S48. pmid:11895154
- 32. Pedersen BK, Tvede N, Klarlund K, Christensen LD, Hansen FR, Galbo H, et al. Indomethacin in vitro and in vivo abolishes post-exercise suppression of natural killer cell activity in peripheral blood. International Journal of Sports Medicine. 1990; 11:127–31. pmid:2338373
- 33. Nieman DC, Henson DA, McAnulty SR, Jin F, Maxwell KR. n-3 polyunsaturated fatty acids do not alter immune and inflammation measures in endurance athletes. International Journal of Sport Nutrition and Exercise Metabolism. 2009; 19:536–46. pmid:19910654
- 34. Skarpańska-Stejnborn A, Pilaczyńska-Szcześniak Ł, Basta P, Foriasz J, Arlet J. Effects of Supplementation with Neptune Krill Oil (Euphasia Superba) on Selected Redox Parameters and Pro-Inflammatory Markers in Athletes during Exhaustive Exercise. Journal of Human Kinetics. 2010; 25:49–57.
- 35. Bloomer R, Larson D, Fisher-Wellman K, Galpin A, Schilling B. Effect of eicosapentaenoic and docosahexaenoic acid on resting and exercise-induced inflammatory and oxidative stress biomarkers: a randomized, placebo controlled, cross-over study. Lipids in Health and Disease. 2009; 8:36. pmid:19691834
- 36. McAnulty SR, Nieman DC, Fox-Rabinovich M, Duran V, McAnulty LS, Henson DA, et al. Effect of n-3 fatty acids and antioxidants on oxidative stress after exercise. Medicine and science in sports and exercise. 2010; 42:1704–11. pmid:20164809
- 37. Rodacki CLN, Rodacki ALF, Pereira G, Naliwaiko K, Coelho I, Pequito D, et al. Fish-oil supplementation enhances the effects of strength training in elderly women. The American Journal of Clinical Nutrition. 2012; 95:428–36. pmid:22218156
- 38. Smith GI, Julliand S, Reeds DN, Sinacore DR, Klein S, Mittendorfer B. Fish oil–derived n–3 PUFA therapy increases muscle mass and function in healthy older adults. The American Journal of Clinical Nutrition. 2015; 102:115–122 pmid:25994567
- 39. Perry C, Pick M, Bdolach N, Hazan-Halevi I, Kay S, Berr I, et al. Endurance exercise diverts the balance between Th17 cells and regulatory T cells. PloS one. 2013; 8:e74722. pmid:24130669
- 40. Gaffen SL. An overview of IL-17 function and signaling. Special Issue—Abstracts and Reviews: 7thJoint Conference of the International Cytokine Society and the International Society for Interferon and Cytokine Research. 2008; 43:402–7.
- 41. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. The Journal of experimental medicine. 1996; 183:2593–603. pmid:8676080
- 42. Iwakura Y, Nakae S, Saijo S, Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens. Immunological reviews. 2008; 226:57–79. pmid:19161416
- 43. Ostrowski K, Rohde T, Asp S, Schjerling P, Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. The Journal of physiology. 1999; 515:287–91. pmid:9925898
- 44. Nieman DC, Johanssen LM, Lee JW, Arabatzis K. Infectious episodes in runners before and after the Los Angeles Marathon. The Journal of sports medicine and physical fitness. 1990; 30:316–28. pmid:2266764