Introduction
There is continued appreciation for the key role of optimised nutrition for maximising adaptation to training stimulus and therefore athletic performance. This has opened the field of ‘performance nutrition’ which has focused primarily on optimal macronutrient consumption and total energy intake. Notwithstanding the foundational role of broader macronutrient optimisation, considerable attention has been given to subgroups of fatty acids such as the omega-3 class of polyunsaturated fatty acids (PUFA). Omega-3 fatty acid supplements, and specifically long-chain omega-3 PUFA (LC n-3 PUFA), are increasingly being recommended within athletic groups and sporting institutes as a potential ergogenic aid, being recognised in 2019 by the National Collegiate Athletic Association (NCAA) as a ‘permissible’ supplement for athletic departments to purchase and provide to athletes(Reference Hosick1) and by Australian Institute of Sport (AIS) in 2021 as a Grade B (emerging scientific support) supplement(2).
The LC n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are the primary LC n-3 PUFA responsible for exerting physiological effects that may benefit physically trained participants. Furthermore, these physiological effects of LC n-3 PUFA supplementation are predominantly mediated through target tissue membrane incorporation of EPA and especially DHA(Reference McLennan3), and the omega-3 index (O3I) (erythrocyte membrane) is a readily accessible marker of membrane composition of other tissues, including skeletal and cardiac muscle. The proportional sum of EPA + DHA within erythrocyte membranes (termed the O3I) represents a marker of habitual LC n-3 PUFA consumption and has been used clinically to stratify risk for cardiovascular disease mortality (<4% high risk, 4–8% moderate risk, >8% low risk)(Reference Harris and Von Schacky4). Currently, mean O3I of large samples of physically trained NCAA athletes from a variety of sports are consistently below 4·5%(Reference Anzalone, Carbuhn and Jones5,Reference Ritz, Rogers and Zabinsky6) , indicating both the potential for supplementation and the need for translation from research into practice and clear LC n-3 PUFA consumption guidelines for athletes. More broadly, these low values of O3I are reflected in other physically trained populations who undergo similar physiological strain as physically trained athletes, such as military personnel, and who may benefit from increased LC n-3 PUFA consumption(Reference Rittenhouse, Scott and Deuster7,Reference Peoples, Larsen and Bowes8) . In addition to the potential for ergogenic benefit from short-term LC n-3 PUFA supplementation, it is important to consider longevity of an athlete’s career, which may be supported by maintaining a protective O3I level long term. It is also worth noting that recent trials highlight the potential application of LC n-3 PUFA supplementation to mitigate the magnitude of head trauma experienced by athletes(Reference Heileson, Anzalone and Carbuhn9–Reference Raikes, Hernandez and Mullins11) and improve recovery from muscle-damaging exercise(Reference Anthony, Macartney and Peoples12,Reference Heileson and Funderburk13) . Thus, prophylactic LC n-3 PUFA supplementation appears to represent a feasible strategy to positively influence the physiology of multiple tissues and, subsequently, enhance functional outcomes.
Whilst the O3I tends to be low in physically trained populations and is readily raised by supplementation(Reference Macartney, Hingley and Brown14), the question remains as to whether any performance benefit can be obtained from having a higher O3I. Several narrative review articles(Reference Thielecke and Blannin15–Reference Murphy and McGlory20) and one systematic review(Reference Lewis, Daniels and Calder21) have attempted to address the question of whether omega-3 supplementation can optimise sport performance or act as an ergogenic aid in physically trained participants. Whilst some reviews suggest a broadly beneficial effect of LC n-3 PUFA supplementation to improve aspects of sport performance(Reference Thielecke and Blannin15,Reference D’Angelo and Madonna16,Reference Philpott, Witard and Galloway19) , others indicate either the evidence is inconclusive(Reference Mickleborough17,Reference Lewis, Daniels and Calder21) or there is no benefit above dietary recommended doses(Reference Murphy and McGlory20). The considerable variability in results across these studies is most often attributed to disparate study design characteristics. However, as none of these reviews has explicitly addressed this topic, it forms the primary outcome of the current review.
The consequence of inappropriate LC n-3 PUFA study design characteristics is most clearly evident in clinical trials with cardiovascular endpoints. Unlike pharmaceutical trials, trials involving LC n-3 PUFA are unique in their inability to entirely eliminate the test substance from dietary consumption and therefore from within tissue membranes at baseline or in any control or placebo group(Reference James, Sullivan and Metcalf22). For example, the contradictory findings in randomised control trials of LC n-3 PUFA supplementation and cardiovascular mortality are largely attributable to the inherent difficulties in creating clear separation between intervention and control groups prior to and following the intervention(Reference McLennan and Pepe23). Within the context of cardiovascular outcomes, factors such as background diet LC n-3 PUFA consumption, measurement of LC n-3 PUFA blood status, LC n-3 PUFA dose and duration have all been identified as key elements which influence the efficacy of trials in cardiac patients(Reference James, Sullivan and Metcalf22,Reference Elagizi, Lavie and O’Keefe24) . Drawing from evidence in this cohort, these study design elements may help guide appropriate LC n-3 PUFA study design creation in healthy individuals with endpoints related to physical performance, muscle recovery or mitigation of neurological consequences related to head trauma injury.
A frequently overlooked consideration in LC n-3 PUFA study design is the prior dietary intake of EPA + DHA through fatty fish, fish oil and other marine sources, which dictate baseline LC n-3 PUFA membrane composition(Reference Browning, Walker and Mander25). This, along with dose, duration and delivery method of EPA + DHA, influences the incorporation of supplemental LC n-3 PUFA within target tissues and may therefore contribute to the mixed conclusions found across review articles. It is currently not possible to ascertain the true benefit, or mechanism by which LC n-3 PUFA are acting within physically trained participants until control of variables known to influence outcome measures is addressed. Therefore, the present study aims to review the methods and methodology of studies supplementing LC n-3 PUFA in physically trained participants to address and correct the issues that might be contributing to the variability in reported outcomes.
Methods
Study overview and inclusion criteria
The primary objective of this systematic literature review was to evaluate the methodological practices in LC n-3 PUFA trials aiming to determine whether LC n-3 PUFA improves physiological performance or recovery metrics in physically trained individuals.
Inclusion criteria were as follows: (a) supplementation with LC n-3 PUFA, regardless of delivery method, dose or duration, (b) inclusion of physically trained participants and (c) measurement of a performance or recovery outcome. Studies were excluded if they met the following criteria: (a) participants were described as physically untrained, (b) study did not provide a description of participant training status to determine if participants were physically trained, (c) study did not include a physical performance outcome or physiological marker related to performance or recovery, (d) study assessed musculoskeletal injury or trauma as a primary outcome and (e) study was a conference paper, book chapter or conference proceeding. Further, studies which met inclusion criteria and (a) shared participant data and (b) included co-ingestion of other nutrients with LC n-3 PUFA were included in the review.
Search parameters and study selection
A systematic search of the literature was conducted in SCOPUS, PubMed and Web of Science electronic databases (from earliest record to April 2022). The search strategy used various combinations of the following keywords: fish* OR krill* OR omega* OR EPA OR eicosapentaenoic* OR DHA OR docosahexaenoic* OR PUFA* AND elite OR professional OR trained OR compet* AND athlet*. Where possible, database results were limited to include only studies involving humans.
The screening process began with the removal of duplicate studies using Endnote, version X8 (Thomson Reuters, Philadelphia, PA, USA). Following this, screening of the title and abstract was performed to remove irrelevant or off-topic studies. The full text of the remaining studies was then retrieved and screened using the inclusion and exclusion criteria. Two authors (G.E.P.: medical and exercise physiologist; R.A.: nutritionist) screened each article, and any discrepancies between the two authors were resolved by consulting a third author (M.J.M.: medical and exercise physiologist).
Assessment of study quality
The Cochrane Collaboration’s Risk of Bias tool for randomised trials version 2 (RoB 2)(Reference Sterne, Savović and Page26) was used to assess the internal validity for each randomised control trial (RoB 2 for RCT) and randomised crossover trial (RoB 2 for crossover trials). Each study is classified as having either low risk, some concerns or high risk for each domain. The internal validity for each non-randomised study was determined by the Risk of Bias in Non-randomised Studies of Interventions (ROBINS-I) tool(Reference Sterne, Hernán and Reeves27). With this tool, each study is classified as having either low risk, moderate risk, serious risk or critical risk for each of seven domains. Two authors (G.E.P.; R.A.) judged the risk of bias, and any discrepancies between the two authors were resolved by consulting a third author (M.J.M.).
To assess the study design, specifically for LC n-3 PUFA interventions, a previously used(Reference Anthony, Macartney and Peoples12) custom five-point quality assessment scale was used on the basis of recommendations from James et al. (Reference James, Sullivan and Metcalf22). Criteria included the following: (i) exclusion of participants with a baseline erythrocyte membrane EPA and/or DHA level above a certain threshold, (ii) whether supplementation resulted in a change in erythrocyte membrane EPA and/or DHA, (iii) exclusion of participants that consumed LC n-3 PUFA supplements, (iv) exclusion of participants consuming more than one fish meal per week, and (v) minimum supplementation duration of 4 weeks. The scale scored each criterion as either satisfied (1 point) or not satisfied (0 points), resulting in a score ranging from 0 to 5.
Results
Studies selected
A total of 369 publications were identified from the literature search of databases, and an additional 3 publications were identified through review of reference lists of identified studies and review articles (Fig. 1). After removal of duplicates, 251 publications were removed on the basis of title and abstract screening. Of the remaining fifty-seven publications, a further eleven were excluded on the basis of full-text screening. Figure 1 outlines the selection process, with reasons for exclusion. A total of forty-six publications met the inclusion criteria and were included in the review (Table 1).
Fig. 1. Prisma flow diagram showing study selection process.
Table 1. Study characteristics and omega-3 study design score
* Estimates based on body mass.
NRS, non-randomised study.
Assessment of study quality
The majority (n = 31) of studies included in this review were randomised control trials (RCTs), and their internal validity was assessed using the Cochrane Collaboration’s RoB 2 tool for RCTs (Figs. 2a and 3a). Five trials were randomised crossover trials and were assessed with the Cochrane Collaboration’s RoB 2 tool for randomised crossover trials (Figs. 2b and 3b). The internal validity of the remaining non-randomised studies (n = 10) was assessed with the Cochrane Collaboration’s ROBINS-I tool (Figs. 2c and 3c).
Fig. 2. Risk of bias graph: review authors’ judgements about each risk of bias item for each included study. Legend. (a) Risk of bias graph generated using Cochrane Risk of Bias 2 tool for RCT studies. (b) Risk of bias graph generated using Cochrane Risk of Bias 2 tool for randomised crossover studies. (c) Risk of bias graph generated using ROBINS-I tool for non-randomised studies.
Fig. 3. Risk of bias summary: review authors’ judgements about each risk of bias item presented as percentages across all included studies. Legend. (a) Risk of bias summary generated using Cochrane Risk of Bias 2 tool for RCT studies. (b) Risk of bias summary generated using Cochrane Risk of Bias 2 tool for randomised crossover studies. (c) Risk of bias summary generated using ROBINS-I tool for non-randomised studies.
Randomised control trials
Most RCTs received an overall risk of bias judgement of ‘some concerns’. This was largely a result of only three studies(Reference Leaf and Rauch28,Reference Lewis, Radonic and Wolever54,Reference Jakeman, Lambrick and Wooley60) providing sufficient detail regarding the randomisation process to warrant a low risk of bias. All studies had some concerns regarding selection of the reported result as no study met the requirements of providing detail of analysis intentions prior to unblinding of outcome data. Those studies which did register details regarding analysis intentions (i.e. through clinicaltrials.gov) often did so after the study was already published. Two studies(Reference Filaire, Massart and Rouveix44,Reference Guzmán, Esteve and Pablos45) received an overall high risk of bias due to providing insufficient information regarding deviations from the intended intervention.
Randomised crossover trials
The five studies included in this review which utilised a randomised crossover design all received an overall high risk of bias. The bias predominantly arose from period and carryover effects. High-risk classification was determined by the lack of confirmation that LC n-3 PUFA blood levels returned to baseline values with a measurement of erythrocyte membrane EPA and/or DHA levels prior to beginning placebo and intervention supplementation. Only one study(Reference Ramos-Campo, Avila-Gandia and Lopez-Roman69) provided sufficient detail regarding participant randomisation to receive low risk for the randomisation process. Mickleborough et al. (Reference Mickleborough, Murray and Ionescu31) and Bunn et al. (Reference Bunn, Crossley and Timiney62) were the only studies which satisfied criteria required for a low risk classification in deviations from the intended intervention.
Non-randomised studies
Of the ten non-randomised studies, there were some concerns in five of those studies, whereas four studies were assessed as having an overall high risk of bias. One study(Reference Buonocore, Verri and Giolitto67) was assessed as having an overall critical risk of bias. The primary concern in the studies that were assessed as having an overall high risk and critical risk was bias due to confounding. Buonocore et al. (Reference Buonocore, Verri and Giolitto67) received a critical risk for this domain as there was no control over baseline measures, nor was there a direct control group.
Assessment of LC n-3 PUFA study designs
The mean LC n-3 PUFA study design score across all forty-six studies was 1·48 ± 1·01 (± SD) (out of a potential 5) with a range of 0 to 4 (Table 1). There were only three studies that achieved a score of 4(Reference Macartney, Hingley and Brown14,Reference Hingley, Macartney and Brown59,Reference Drobnic, Storsve and Burri71) . One study excluded participants with a baseline erythrocyte membrane EPA and/or DHA level above a set threshold(Reference Drobnic, Storsve and Burri71); however, no study successfully met all the criteria for a score of 5.
LC n-3 PUFA dose, duration and delivery method
A combination of EPA + DHA was predominantly used (n = 38) usually as a fish oil or concentrated fish oil, with one study using isolated EPA and seven studies using isolated DHA. Of the thirty-eight studies that used a combined EPA + DHA supplement, twenty-four studies used an EPA dominant capsule, nine studies used a DHA dominant capsule, four studies used an equal EPA + DHA ratio and one study did not state the EPA + DHA content. In the forty-five studies that reported the component doses of EPA and DHA separately, the average EPA dose was 1242 mg/d (±1284 SD; min, 120 mg/d; max, 6000 mg/d) and the average DHA dose was 978 mg/d (±765 SD; min, 96 mg/d; max, 3500 mg/d). Supplementation duration ranged from 1 d (acute) to 84 d, with an average of 40 d (±20 SD).
Supplemental LC n-3 PUFA delivery was achieved through capsules in 67% of studies (n = 31) and as a beverage in 22% (n = 10) of studies. The remaining studies used pills (n = 1), used chewable supplements (n = 2) or did not state delivery method (n = 2). Authors identified the source of LC n-3 PUFA as fish-derived in 61% of studies (n = 28), whereas 26% of studies (n = 12) did not state the origin of LC n-3 PUFA. The remaining studies used krill oil (n = 4), algal oil (n = 1) or seal oil (n = 1).
Measurement of LC n-3 PUFA blood status
Overall, 54% of studies (n = 25) included a biological measure of LC n-3 PUFA status. Erythrocyte membrane LC n-3 PUFA was predominantly chosen as the biological marker in those studies (n = 12), with LC n-3 PUFA of blood plasma measured in six studies (n = 6), whole blood in five (n = 5) and neutrophils in two (n = 2).
Dietary assessment
A form of dietary assessment was conducted in 48% of studies (n = 22). Most studies used food records (n = 16) ranging from 1 d to 7 d. Other forms of dietary assessment included food frequency questionnaire (n = 4), or food recall (n = 1), with one study omitting description of the tool used for dietary assessment. One study(Reference Philpott, Bootsma and Rodriguez-Sanchez65) provided all food items and fluids to participants with the intent to create an energy deficit to induce weight loss. In the twenty-two (n = 22) studies that measured dietary intake, sixteen (n = 16) studies reported no difference in major nutrients or energy intake between groups at baseline; however, only six (n = 6) of those studies reported no difference in baseline and post-supplement dietary assessments. The remaining six (n = 6) studies did not provide dietary data or sufficient detail to determine if there were differences in dietary intake between groups or after supplementation.
Discussion
This review has identified a shortfall in design considerations within studies that have investigated the effects of LC n-3 PUFA in physically trained populations. Firstly, the risk of overall study bias, as classified via the Cochrane Collaboration’s and ROBINS-I tools, was extraordinarily high, with the majority of studies (65%) identified as ‘some concerns’, the remaining studies (35%) categorised as ‘high risk’ and no studies achieving ‘low risk’ (Fig. 2). Across studies, the risk of bias was often attributed to critical flaws in the design, conduct, analysis and reporting of outcomes. Most importantly, serious concern should be drawn from the lack of appreciation for designing and implementing studies that adhere to principles for interrogating the specific effects of LC n-3 PUFA including (i) measurement of LC n-3 PUFA tissue status, (ii) assessment of background diet, (iii) baseline LC n-3 PUFA biomarker informed exclusion criteria and (iv) the interaction of LC n-3 PUFA dose and duration. Such widespread lack of adherence to fundamental study design considerations explain why it has been difficult to either draw conclusions relating to the benefits or detriments of LC n-3 PUFA supplementation in physically trained populations, or make recommendations for applied practice.
Measurement of LC n-3 PUFA tissue status
The physiological effects of LC n-3 PUFA are predominately governed by their relative contribution to and, following supplementation, their alteration of cell membrane fatty acid proportional composition within the target tissue(Reference McLennan3,Reference Calder73–Reference McLennan, Owen and Slee75) . Many studies examined in this review sought to determine the effects of LC n-3 PUFA on outcome measures related to improved cardiovascular endurance and aerobic capacity, physical strength and power, and the attenuation of exercise-induced inflammatory and muscle damage processes. Often the emphasis is placed on cardiac and skeletal muscle as the primary tissues where improvement is likely to be actualised in outcome measures. Measurement of LC n-3 PUFA at these target tissues would provide evidence that the LC n-3 PUFA intervention was successful in altering target tissue cell membrane fatty acids and therefore give confidence of a causal relationship between LC n-3 PUFA supplementation and outcome measures. In the case of LC n-3 PUFA status in skeletal muscle, a muscle biopsy would represent the gold standard for establishing LC n-3 PUFA incorporation within the target tissue. Whilst this has been undertaken previously(Reference McGlory, Wardle and Macnaughton76–Reference Smith, Atherton and Reeds78), there remain limitations around participant burden, cost and researcher skill. Furthermore, animal studies reveal systematic variations according to muscle fibre type analysed(Reference Macartney, Peoples and Treweek79), therefore raising questions of choice or access to muscles that correlate with human physiological or functional outcomes.
If a skeletal muscle biopsy is not practical, or if cardiac muscle is the target tissue, an appropriate blood biomarker that is reflective of LC n-3 PUFA incorporation in cardiac and skeletal muscle cell membranes should be used as a surrogate to indicate target tissue cell membrane LC n-3 PUFA status. The selection of an appropriate blood biomarker requires evaluation of temporal changes occurring within the target tissue cell membrane, in parallel with the chosen blood biomarker, following LC n-3 PUFA supplementation. Erythrocyte membranes appear to be the blood biomarker that most appropriately reflects LC n-3 PUFA incorporation into most major organs(Reference Fenton, Gurzell and Davidson80). Changes within erythrocyte membranes follow a delayed incorporation and desaturation curve and have lower biological variability(Reference Harris and Thomas81) than plasma phospholipids(Reference Cao, Schwichtenberg and Hanson82). This is mirrored in skeletal and cardiac muscle, which follow similar incorporation rates to that of erythrocyte membranes(Reference Fenton, Gurzell and Davidson80,Reference Harris, Sands and Windsor83,Reference Metcalf, James and Gibson84) . Relationships between cardiac muscle and blood markers are best demonstrated by simultaneously sampling blood and atrial biopsy during cardiac surgery. Such studies reveal that erythrocyte membrane fatty acid composition provides the best correlate of any blood component with cardiac incorporation(Reference Metcalf, Cleland and Gibson85). Furthermore, slower fluctuations in incorporation and washout, plus a nearer to 1:1 relationship between erythrocyte and atria show erythrocytes to be a better long-term marker of muscle incorporation, compared with plasma. In the present review, an appropriate blood biomarker, namely erythrocyte EPA, DHA or EPA + DHA, was collected in only 28% of studies, and within the studies that did measure erythrocyte membrane fatty acids, few established marked changes from baseline.
Few studies consider the washout characteristics of LC n-3 PUFA from cardiac muscle or skeletal muscle cell membranes following cessation of LC n-3 PUFA supplementation. However, their slower incorporation rate would indicate that clearance of LC n-3 PUFA would follow a delayed, rather than expedient time course. Fatty acid washout is much slower from erythrocyte membranes than plasma(Reference Cao, Schwichtenberg and Hanson82). Indeed, LC n-3 PUFA, especially DHA, can remain elevated in erythrocytes beyond 18 weeks after cessation of fish or fish oil supplementation for 6 weeks(Reference Brown, Pang and Roberts86) and for as long as 6 months(Reference Katan, Deslypere and van Birgelen87) after 12 months supplementation. This raises concern in the four studies that used a crossover design with a washout period ranging from 2 weeks to a maximum of 4 weeks. Cao et al. (Reference Cao, Schwichtenberg and Hanson82) has demonstrated that 4 weeks after cessation of LC n-3 PUFA supplementation, erythrocyte membrane EPA + DHA remained statistically higher than baseline and did not entirely return to baseline levels until 16 weeks after supplement cessation. If a crossover study design is to be used, a measurement of erythrocyte membrane EPA + DHA should be collected before and after each supplement block to confirm LC n-3 PUFA levels have returned to baseline during the washout period, prior to crossing over to the alternate supplement.
Background diet LC n-3 PUFA
As has been explored by James et al.(Reference James, Sullivan and Metcalf22), intervention trials involving LC n-3 PUFA are unique when compared with pharmacological drug trials. In drug trials it is common, possible and indeed essential to eliminate consumers of the intervention drug entirely from the control group. LC n-3 PUFA are found commonly within foods consumed on a semi-regular basis, and whilst population intakes are below desirable targets(Reference Stark, Van Elswyk and Higgins88,Reference Meyer89) , their presence in food and within the body should be acknowledged. The identification and therefore exclusion of participants who consumed one or more fish meals per week was conducted in only four of the forty-six studies in this review. More studies (n = 19) considered supplementation and established criteria for exclusion for those taking fish oil supplements, yet only five of those studies measured a biomarker of habitual LC n-3 PUFA consumption (erythrocyte membrane EPA + DHA). Without exclusion parameters, the often wide ranges in LC n−3 PUFA levels at baseline and subsequent failure to avoid overlap between treatment and control groups after treatment were identified as a common flaw in randomised control trials of cardiovascular outcomes, preventing valid assessments of outcomes and conclusions(Reference McLennan and Pepe23).
Broad dietary intakes involving total energy and macronutrients have a profound effect on sport performance, particularly in athletes undergoing physiologically stressful exercise(Reference Thomas, Erdman and Burke90). Standardisation and monitoring of dietary intake is recognised as an important component in trials assessing performance related outcomes in physically trained participants(Reference Jeacocke and Burke91). Whilst standardisation of dietary intake between trials would not be possible due to the variance of dietary requirements between individuals and sports, it should be considered and accounted for within individual trials. As a minimum, measuring dietary intake at baseline and post-intervention limits the confounding of key variables known to influence performance. The selection of an appropriate dietary assessment tool for use within physically trained populations is outside the scope of this review(Reference Magkos and Yannakoulia92,Reference Capling, Beck and Gifford93) ; however, regardless of the dietary assessment tool used, only six studies in the current review reported no change in dietary intake at baseline and post-intervention periods. Considering these trials sought to manipulate diet through LC n-3 PUFA supplementation, the general lack of dietary assessment data and the heterogeneous approaches taken to assess and report on dietary intake is surprising. Nevertheless, with respect to LC n-3 PUFA, dietary assessment cannot match the accuracy and reliability of a blood biomarker such as O3I, due to the difficulty in capturing the infrequent consumption pattern of fatty fish(Reference Serra-Majem, Nissensohn and Øverby94).
Baseline exclusion
The LC n-3 PUFA enrichment of erythrocyte fatty acid membranes through dietary feeding of LC n-3 PUFA is largely a function of current erythrocyte levels, whereby a higher erythrocyte EPA + DHA level is less responsive to further increases(Reference Cao, Schwichtenberg and Hanson82). It is therefore paramount to start all participants at an equal and low erythrocyte membrane EPA + DHA level if LC n-3 PUFA dosage and duration are fixed. This is particularly important considering that some physiological effects may be derived from achievable dietary intakes(Reference Macartney, Hingley and Brown14,Reference Hingley, Macartney and Brown59) rather than pharmacological doses as described for cardiovascular effects(Reference McLennan3). None of the studies in this review established predefined criteria for exclusion based on erythrocyte membrane EPA and/or DHA. The precise value of erythrocyte membrane EPA + DHA to be used as a criterion for the purpose of athletic benefit has not been established. The O3I (erythrocyte EPA + DHA expressed as a percentage of total fatty acids) has predominantly been used to categorise risk associated with cardiovascular mortality(Reference Harris and Von Schacky4), whereby those with a value below 4% are associated with the highest risk, those between 4% and 8% associated with intermediate risk and those above 8% associated with the lowest risk. An O3I below 6·5% was arbitrarily chosen as a criterion for baseline participant exclusion in trials investigating incorporation of EPA and DHA within blood in both short(Reference Fisk, Kindberg and Hustvedt95) and long-term studies(Reference West, Kindberg and Hustvedt96), and James et al. (Reference James, Sullivan and Metcalf22) demonstrated in a cardiovascular RCT that this would likely give satisfactory separation of treated and control groups. However, it may be appropriate to use statistical methods, such as exclusion of the 90th percentile of O3I, until further research clarifies the optimal ranges for athletic benefit.
Separation of groups and the interaction of LC n-3 PUFA dose and duration
Upon establishment of homogeneous and low LC n-3 PUFA tissue status at baseline, the proposed LC n-3 PUFA intervention must be assessed for its ability to create clear group separation post-supplementation. This is a function of both the dosage of EPA and DHA, in addition to supplement duration. Browning et al.(Reference Browning, Walker and Mander25) explored this in their paper on incorporation of varying doses of EPA + DHA over a lengthy duration of 12 months. Whilst moderate doses (approximately two portions of fish per week) were able to establish a similar maximal erythrocyte membrane EPA and DHA incorporation compared with higher doses (approximately four portions of fish per week), the time to maximal incorporation was more than double that of the higher dose. Furthermore, the objectives of the study and primary mechanism of LC n-3 PUFA should also be considered when assessing optimal dose and duration. Higher doses (>2 g/d EPA + DHA) which favour EPA over DHA are often chosen in studies in which LC n-3 PUFA supplementation is found to influence muscle mass(Reference Smith, Julliand and Reeds97), strength(Reference Smith, Julliand and Reeds97,Reference Dalle, Van Roie and Hiroux98) and muscle protein metabolism(Reference Smith, Atherton and Reeds99,Reference Yoshino, Smith and Kelly100) in older adults. The implementation of higher doses should be taken with caution as the mechanism by which LC n-3 PUFA exert effects on muscle protein metabolism is unclear and therefore dose and duration require further investigation.
The present review has highlighted a broad range of EPA + DHA doses (min: 300 mg/d; max: 6400 mg/d), with supplement duration ranging from 1 d to 84 d. With supplement dose and duration being linked to incorporation within various lipid pools and therefore physiological effects, it was expected that a negative association between dose and duration would be present across studies in this review. In fact, there was no association between selection of EPA + DHA dose and supplement duration (Fig. 4), nor substantial reasoning for either. Whilst a statistically significant increase was reported in all studies that measured erythrocyte membrane EPA and/or DHA, there were clear differences in the magnitude of group separation. Zebrowska et al.(Reference Zebrowska, Hall and Stolecka-Warzecha72) reported a significant increase in O3I following LC n-3 PUFA intervention, both within group and between groups post-supplement; however, O3I was increased by only 0·9%, reaching a final mean value of 4·8% (versus 3·9% in the control group). Conversely, group separation was more clearly defined by others(Reference Oostenbrug, Mensink and Hardeman29,Reference Malaguti, Baldini and Angeloni33,Reference Buckley, Burgess and Murphy35) , with increases in O3I ranging from 2% to >4%, with no change from baseline in the relatively low O3I of the control groups.
Fig. 4. Correlation between supplement dose and supplement duration for included studies (n = 46).
Conclusion and future recommendations
LC n-3 PUFA has recently gained substantial interest from sport scientists and researchers aiming to improve athletic outcomes. This is evident from more than half of the research papers in this review being conducted within the past 10 years. Nevertheless, despite this research intensity, the potential benefits of LC n-3 PUFA remain inconclusive. The current review has revealed many contributary design and measurement inconsistencies. First, a clear mechanistic hypothesis for LC n-3 PUFA was rarely articulated, resulting in substantial variation of dose and duration across individual studies. Second, inadequate assessment of broader background dietary intake and background dietary LC n-3 PUFA consumption. Finally, the paucity of appropriate LC n-3 PUFA blood biomarker assessment, leading to uncertainty in intervention efficacy. This highlights the need for widespread implementation of the LC n-3 PUFA study design considerations outlined throughout this review (shown graphically in Fig. 5) as a priority for future research in physically trained individuals.
Fig. 5. Study design considerations for LC n-3 PUFA trials in physically trained individuals.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflict of interest
None.
Authorship
All the authors have contributed to the manuscript and approved the final submission. Study design, data collection and data analysis were carried out by R.A., G.E.P. and M.J.M. Data interpretation and manuscript preparation were undertaken by all authors.
