Effects of eight weeks of eicosapentaenoic acid and medium-chain triacylglycerol structured lipid intake on EPA/AA ratio and muscle performance in young men

Abstract

Background

Structured triglycerides (STGs), in which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are esterified with medium-chain triglycerides (MCTs), have demonstrated greater bioavailability and potential benefits in improving endurance and reducing post-exercise strength loss compared with physical mixtures (PMs) of EPA and MCTs. However, it remains unclear whether STGs have superior effects on blood EPA levels and muscular endurance and fatigue. This study compared the effects of 8-week STG and PM intake on blood EPA levels, muscular endurance, and fatigue following resistance exercise.

Methods

Twenty-eight healthy young men were randomly assigned to an STG group (n = 15) or a PM group (n = 13) in a double-blind, parallel-group, active comparator trial. Participants consumed 4,560 mg/day of the test oil (600 mg EPA, 260 mg DHA) for 8 weeks. After the intervention, the participants performed four sets of leg extensions to exhaustion at 40% of their body weight. Muscular endurance was assessed by the number of repetitions, and fatigue was evaluated by changes in maximal voluntary contraction, range of motion, thigh circumference, muscle thickness, echo intensity, and jump performance.

Results

The STG group showed a significantly greater increase in the serum EPA/arachidonic acid (AA) ratio compared with the PM group. However, no significant differences were found between groups in repetition counts or fatigue-related measures.

Conclusion

Eight weeks of STG supplementation improved the blood EPA/AA ratio more than a PM, but did not yield superior effects on muscle endurance or fatigue.

1. Introduction

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both of which are found in fish oil, have been suggested to enhance cardiorespiratory endurance capacity [1] owing to their potential to improve red blood cell deformability and promote lipid oxidation during exercise [2,3]. In addition, several studies have reported the effects of EPA and DHA on local muscular endurance and fatigue during resistance exercise [4–6].

In our previous study, healthy young men ingested 600 mg of EPA and 260 mg of DHA daily for 8 weeks [4]. The participants performed a muscular endurance test comprising six sets of five isokinetic elbow flexion repetitions. The group supplemented with EPA/DHA exhibited attenuated declines in muscular endurance performance and suppressed reductions in muscle strength immediately after exercise compared to the placebo group [4]. However, in another study involving healthy young men who consumed 600 mg of EPA and 260 mg of DHA daily for 8 weeks and performed leg extensions to exhaustion, no significant improvements were observed in muscular endurance performance or attenuation of exercise-induced muscle fatigue [5]. Therefore, although EPA/DHA supplementation appears to effectively enhance muscular endurance performance, its efficacy has not been demonstrated in exhaustive resistance exercise involving the quadriceps muscles.

The EPA/AA ratio is the proportion of EPA to arachidonic acid (AA). It reflects the balance of omega-3 and omega-6 polyunsaturated fatty acids that shape eicosanoid signaling and inflammation. A higher EPA level relative to AA is associated with a less pro-inflammatory eicosanoid profile and may support oxidative metabolism and vascular function during exercise [7]. Therefore, we consider the EPA/AA ratio to be a primary endpoint in studies of the effects of exercise, together with serum EPA, alongside task-specific measures of muscular endurance and fatigue.

Structured triacylglycerols (STGs) are molecularly re-esterified lipids in which EPA and DHA are chemically bound to the glycerol backbone together with medium-chain triglycerides (MCTs). In contrast, physical mixtures (PMs) are simple oil blends in which EPA, DHA, and MCTs are present but are not incorporated within the same glycerol backbone. This structural difference may influence the digestion, absorption, and bioavailability of the fatty acids. Furthermore, EPA and AA compete for the same enzymes—cyclooxygenase (COX) and lipoxygenase (LOX) [8]—so a relative increase in EPA shifts the profile toward anti-inflammation by relatively suppressing the production of pro-inflammatory eicosanoids (prostaglandin E₂, leukotriene B₄, thromboxane A₂) [9]. Consequently, the EPA/AA ratio is widely referenced as an indicator of inflammation and cardiovascular risk [10]. In the sports domain, it is also expected to be applied as a biomarker for managing excessive inflammation and delayed-onset muscle soreness [11], maintaining endothelial function and oxygen utilization efficiency in endurance events, and monitoring the optimization of training load and recovery strategies [12]. Increases in the EPA/AA ratio accompanying EPA intake are therefore suggested to be associated with reduced risk during exercise and improved conditioning.

Our recent study demonstrated that supplementation with either PM oil or oil in which EPA/DHA and MCTs are incorporated within the same glycerol backbone influenced cardiorespiratory endurance capacity [13]. Specifically, the time to reach the anaerobic threshold during a cycling exercise was significantly prolonged after supplementation in the STG group [13]. Furthermore, the time to exhaustion during the cycling exercise was also significantly longer in the STG group. These findings suggest that STG supplementation enhances endurance performance during cycling [13]. In addition, we have recently investigated the effects of STG supplementation on quadriceps muscular endurance and fatigue during leg extensions [6]. Our findings demonstrated that increases in serum EPA concentrations and the EPA/AA ratio following STG supplementation were associated with improved muscular endurance performance and reduced exercise-induced muscle fatigue [6]. However, this study comprised only a two-group comparison between STG and placebo, and no study has directly compared the effects of STG and PM supplementation on changes in serum EPA concentrations and the EPA/AA ratio. Moreover, differences in their effects on muscular endurance performance have not yet been examined.

Therefore, in the present study, we 1) compared the changes in the serum EPA concentration and EPA/AA ratio following 8 weeks of supplementation with either STG or PM, and 2) investigated the effects of STG and PM supplementation on muscular endurance and fatigue during leg extensions. We hypothesized that STGs would cause greater increases in serum EPA and the EPA/AA ratio than a PM and that these biochemical differences might lead to better muscular endurance and attenuated fatigue under the present protocol.

2. Materials and methods

2.1. Participants

A total of 28 healthy untrained men (age, 19.4 ± 1.3 years; height, 171.9 ± 5.0 cm; weight, 68.4 ± 9.6 kg; body mass index [BMI], 23.1 ± 2.7) were recruited for this study. Female participants were excluded from the present study to eliminate the potential influence of hormonal fluctuations associated with the menstrual cycle. None had any food allergies. In addition, the enrolled participants had not participated in any regular resistance training for at least 1 year before the experiment. They were requested to avoid participation in other clinical trials and interventions such as massage, stretching, strenuous exercise, excessive consumption of food (including omega-3 and omega-6 polyunsaturated fatty acids) or alcohol, and the use of any supplements or medications during the experimental period. All participants were provided with detailed explanations of the study protocol before participation and signed an informed consent form adhering to the tenets of the Declaration of Helsinki before being enrolled in this study. The study was approved by the Ethics Committee for Human Experiments at Teikyo Heisei University (ID: 2023-134) and registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR identifier: UMIN000054425).

2.2. Study design

This double-blind, parallel-group, active comparator trial was designed based on our previous studies [4,5]. All participants were randomly assigned to two groups to minimize the inter-group differences in age, body weight, and BMI. They ingested either EPA/MCT structured lipids (STG group, n = 15; age, 19.7 ± 1.2 years; height, 172.0 ± 4.9 cm; weight, 67.7 ± 9.2 kg; BMI, 22.9 ± 3.1) or EPA/MCT mixed in oil (PM group, n = 13; age, 19.0 ± 1.4 years; height, 171.8 ± 5.4 cm; weight, 69.2 ± 10.3 kg; BMI, 23.4 ± 2.3) for 8 weeks before the exercise experiment. During the 8-week supplementation period, no exercise intervention was implemented, and participants were instructed to avoid initiating any training programs and to maintain their usual daily living activities. Nutritional status was assessed in all participants before and after (immediately before exercise) the 8-week supplementation using a food frequency questionnaire based on food groups (FFQg, version 3.5; Kenpakusha, Tokyo, Japan) [14,15]. Sequence allocation concealment and blinding to participants and representatives were maintained throughout the experimental period. At the end of the study, we checked the subjective day-to-day records and pill count. To improve the reliability of the pill count, participants were provided with an excess number of pills and instructed to return any remaining pills at the end of the study. In addition, we measured serum AA, dihomo-gamma-linolenic acid (DGLA), EPA, DHA, and EPA/AA in blood samples before and after the 8-week supplementation.

2.3. Supplements

The STG group consumed interesterified EPA/MCT STGs, while the PM group consumed EPA/MCT mixed in oil. Both groups consumed 10 soft capsules (Nissui Corporation, Tokyo, Japan) per day for 8 weeks, and the total consumption was 4,560 mg per day (600 mg EPA and 260 mg DHA). Participants ingested the supplements with water 30 min after meals. The STG used in this study are designer lipids in which EPA/DHA and medium-chain fatty acids (MCFA) are re-esterified onto the same glycerol backbone, and after ingestion they are digested and absorbed as a single TAG molecule. In contrast, the PM is a simple blend of an EPA/DHA-containing oil and an MCT oil without molecular-level coupling, and STG have been shown to enhance bioavailability of EPA/DHA compared with PM [16].

2.4. Blood samples

Participants fasted for 8 h before blood samples were obtained from the forearm. Blood samples were allowed to clot at room temperature (23 °C) and were then centrifuged at 3,000 rpm for 10 min at 4 °C. The serum was extracted and stored at −20 °C until analysis. Serum levels of AA, DGLA, EPA, DHA, and EPA/AA were measured using gas chromatography. Participants did not take supplements prior to blood collection after the intervention.

2.5. Experimental procedure

All tests were performed in a temperature-controlled room maintained at 23 °C. Upon arrival, baseline maximal voluntary contraction (MVC) torque, blood lactate, knee joint range of motion (ROM), thigh circumference, muscle thickness, echo intensity, and jump performance were assessed before the muscular endurance test. After the baseline measurements had been made, participants performed a muscular endurance test. After the test, participants repeated the MVC torque, blood lactate, ROM, thigh circumference, muscle thickness, echo intensity, and jump performance tests.

2.6. Muscular endurance test

The muscular endurance test comprised a knee extensor load with weights equal to 40% of the participant's body weight. The protocol was based on previous studies [5,17]. The participants performed repeated bilateral knee extensions at 0.5-Hz frequency (metronome-assisted) for four sets in total. Each set was terminated when the participant failed to maintain adequate extensions. The resting periods between sets were 20, 30, and 40 s, respectively.

2.7. Maximal voluntary isometric contraction torque

After a warm-up comprising three to five knee extensions, MVC torque during isometric knee extension of the dominant leg was measured using a dynamometer (System 4 Pro, Biodex Medical Systems, Inc., Shirley, NY) at a knee joint angle of 90° flexion [18,19]. Following sufficient familiarization and warm-up, participants performed three 3-s MVC trials with a 180-s rest between trials. The MVC was considered to be the peak torque.

2.8. Blood lactate

Blood lactate concentrations were measured with a portable Lactate-Pro analyzer (Arkray Inc., Kyoto, Japan) before and immediately after exercise using fingertip blood samples [20].

2.9. Knee joint ROM

The knee ROM was determined as the difference in joint angles between maximal voluntary flexion and extension of the knee joint using a goniometer (Takase Medical, Tokyo, Japan). Flexion was measured when the participant attempted to maximally flex the knee joint of the exercised leg to touch his hip with his heel while keeping the knee joint aligned with the standing leg and supporting the body by placing a hand on a wall 30 cm from the foot. Extension was measured when the participant attempted to extend the knee joint of the exercised leg as much as possible. The ROM was calculated by subtracting the flexion from the extension of the knee joint [21,22].

2.10. Thigh circumference

The thigh circumference was measured using a tape measure. With each participant standing with his feet approximately 10 cm apart and with his body weight evenly distributed on both feet, we measured the perimeter distance of the thigh perpendicular to the long axis of the femur at the marked mid-trochanterion-tibiale level [23]. The measurements were performed three times at each time point, and the average of the three measurements was used for further analysis.

2.11. Muscle thickness and echo intensity

B-mode ultrasound images of the vastus lateralis, rectus femoris, and vastus medialis muscles were captured using an ultrasound device (SONIMAGE HS1, Konica Minolta, Tokyo, Japan). For image acquisition, the probe was positioned as follows: for the vastus lateralis and rectus femoris, at the level of the lateral femoral epicondyle and 50% of the distance to the greater trochanter; for the vastus medialis, at the lateral femoral epicondyle and 30% distal to the greater trochanter. The same gain and contrast were used throughout the experimental period. Transverse images of each muscle were transferred to a computer as bitmap files (.bmp) and analyzed. The thicknesses of the vastus lateralis, rectus femoris, and vastus medialis were manually calculated by tracing using image analysis software (ImageJ, National Institutes of Health, Bethesda, MD, USA). The mean muscle echo intensity of the region of interest (20 × 20 mm) was calculated using the same software to generate a grayscale histogram (0, black; 100, white) for the region, as described in previous studies [24,25].

2.12. Jump performance

Jump performance during squat jump and countermovement jump was assessed. The participants performed each jump on a jump mat (Multi Jump Tester II, DKH Inc., Tokyo, Japan) connected to a computer, with instructions to jump as high as possible. The flight and contact times were recorded during the jump, and the jump height was calculated based on the flight time using the following formula: jump height (cm) = 1/8 (flight time) × gravitational acceleration (=9.81 m/s²). The squat jump and countermovement jump tests were performed twice each, with a 2-minute rest between tests. The highest jump height for each participant was used in the analysis [25].

2.13. Statistical analysis

All analyses were performed using SPSS software version 28.0 (IBM Corp., Armonk, NY). Values are expressed as means ± standard deviations. Changes in the dependent variables (blood samples, repetitions during muscular endurance testing, MVC torque, blood lactate, knee ROM, jump performance, circumference, echo intensity, and muscle thickness) over time were compared between the STG and PM groups using a two-way repeated measures analysis of variance. When a significant interaction effect was detected, we performed Bonferroni multiple comparisons as a post hoc test. A t-test was performed to compare the changes in EPA and the EPA/AA ratio before and after supplementation in the two groups. In addition, a Student's t-test was applied to compare the groups in terms of the number of total repetitions of the endurance test. Pearson's correlation coefficient (r < 0.1 indicating for no correlation, 0.1–0.3 for weak correlation, 0.3–0.6 for moderate correlation, >0.6 for strong correlation) was used to assess the relationships between the increase in EPA, DHA, and the EPA/AA ratio before and after supplementation and the percent reduction in the number of repetitions from the first to fourth sets. A p-value of <0.05 was considered statistically significant.

3. Results

No significant differences were observed between the STG and PM groups in age, height, body weight, and BMI. In addition, the food frequency questionnaire revealed that there were no significant differences in energy intake and consumption of protein, fat, carbohydrate, and omega-3 polyunsaturated fatty acids between the groups during the intervention period. Furthermore, as shown in Table 1, no significant interaction effects were observed in the two groups before and after the 8-week supplementation in terms of AA, DGLA, EPA, and DHA levels. On the other hand, the interaction was significant for the EPA/AA ratio (p < 0.05), and both groups showed a significant increase after compared to before (p < 0.05). However, the difference between the groups after supplementation was not significant.

Table 1.

Changes in serum AA, dihomo-gamma-linolenic acid, EPA, DHA, and EPA/AA before and after 8-week supplementation.

		PM group			STG group
AA (μg/mL)	Before supplementation	197.8 ± 54.8			200.7 ± 62.2
	After supplementation	186.6 ± 35.6			175.1 ± 51.1
Dihomo-gamma-linolenic acid (μg/mL)	Before supplementation	37.1 ± 10.7			37.6 ± 17.7
	After supplementation	33.9 ± 9.1			30.7 ± 13.4
EPA (μg/mL)	Before supplementation	23.2 ± 15.5			19.5 ± 13.6
	After supplementation	41.7 ± 26.5			52.9 ± 23.5
DHA (μg/mL)	Before supplementation	67.0 ± 25.3			66.8 ± 31.4
	After supplementation	74.4 ± 32.7			74.2 ± 18.8
EPA/AA	Before supplementation	0.125 ± 0.085			0.101 ± 0.072
	After supplementation	0.223 ± 0.127†			0.334 ± 0.203†

AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; PM, physical mixture; STG, structured triglyceride.

^† p < 0.05 for the difference from the before supplementation value.

In the between-group comparison of the changes before and after supplementation, the increase in blood EPA concentrations tended to be greater in the STG group than in the PM group (p = 0.06, Figure 1A). Furthermore, the EPA/AA ratio showed a significantly greater increase in the STG group compared to the PM group (p < 0.05, Figure 1B).

Figure 1.

Comparison of changes in the blood EPA concentration (A) and EPA/AA ratio (B) before and after 8 weeks of supplementation. Data are expressed as the mean ± standard deviation. AA, arachidonic acid; EPA, eicosapentaenoic acid.

During the muscular endurance test, the number of repetitions decreased significantly from the first to the fourth set in both groups, but there was no difference between the groups (Figure 2A). In addition, there were no significant differences in the total number of repetitions (four sets) between the groups (Figure 2B). Blood lactate increased immediately after exercise in both the STG and PM groups (p < 0.05, Figure 2C), with no significant difference between the two groups.

Figure 2.

Repetitions to exhaustion for each set (A) and the total number of repetitions (B) during the muscular endurance test in the PM and STG groups. Changes in blood lactate in the PM and STG groups before and immediately after exercise (C). Data are expressed as the mean ± standard deviation. PM, physical mixture; n.s., not significant; STG, structured triglyceride.

No correlation was observed between the differences in blood EPA concentrations before and after supplement intake and the muscular endurance test, the number of repetitions from the first to fourth set (one set, r = −0.14, p = 0.46; two sets, r = −0.07, p = 0.73; three sets, r = −0.04, p = 0.83; four sets, r = −0.02, p = 0.90), the total number of repetitions (four sets) (r = −0.10, p = 0.60), and the percentage change (r = −0.33, p = 0.12). Similarly, no correlation was observed between the differences in the EPA/AA ratio and the muscular endurance test, the number of repetitions from the first to fourth set (one set, r = −0.18, p = 0.37; two sets, r = −0.09, p = 0.64; three sets, r = −0.06, p = 0.75; four sets, r = −0.06, p = 0.78), the total number of repetitions (four sets) (r = −0.14, p = 0.49), and the percentage change (r = −0.36, p = 0.06).

As shown in Table 2, no significant interactions were observed for MVC, knee ROM, circumference, muscle thickness, echo intensity, squat jump, and countermovement jump. The main effect of time was significant in both the PM and STG groups (p < 0.05), except for ROM, but there were no significant differences between the two groups.

Table 2.

Changes in MVC, ROM, circumference, muscle thickness, echo intensity, squat jump, and countermovement jump before and after muscular endurance testing.

				Two-way ANOVA
		PM group	STG group	Interaction	Time	Group
MVC torque (Nm)	Before	274.0 ± 70.2	262.2 ± 38.9	p = 0.84	p = 0.00	p = 0.84
	After	232.2 ± 71.6	217.1 ± 54.5
ROM (deg)	Before	115.8 ± 12.7	121.2 ± 8.3	p = 0.75	p = 0.06	p = 0.19
	After	114.0 ± 11.2	118.7 ± 9.3
Circumference (cm)	Before	56.0 ± 4.6	53.8 ± 5.3	p = 0.73	p = 0.00	p = 0.22
	After	56.3 ± 3.9	54.4 ± 4.6
Muscle thickness (mm)	Before	93.2 ± 8.9	95.2 ± 9.5	p = 0.12	p = 0.00	p = 0.38
	After	104.7 ± 8.4	101.9 ± 8.5
Echo intensity (AU)	Before	10.3 ± 1.1	9.8 ± 1.0	p = 0.18	p = 0.00	p = 0.87
	After	10.7 ± 0.9	10.6 ± 1.2
Squat jump (cm)	Before	35.4 ± 4.3	31.5 ± 5.4	p = 0.76	p = 0.00	p = 0.19
	After	33.6 ± 4.1	29.2 ± 4.7
Countermovement jump (cm)	Before	39.5 ± 5.2	37.4 ± 4.5	p = 0.73	p = 0.00	p = 0.22
	After	35.2 ± 6.4	32.5 ± 5.5

ANOVA, analysis of variance; AU, arbitrary unit; MVC, maximal voluntary contraction; PM, physical mixture; ROM, range of motion; STG, structured triglyceride.

4. Discussion

In this study, we compared the effects of 8 weeks of supplementation with EPA/MCT STGs and an EPA/MCT PM on changes in the serum EPA concentration and EPA/AA ratio. In addition, we examined their influence on muscular endurance performance and fatigue during leg extensions. The results showed that STG supplementation tended to produce a greater increase in the serum EPA concentration than PM and led to a significant increase in the EPA/AA ratio. In contrast, no significant differences between groups were observed for muscular endurance performance during exercise or muscle fatigue following exercise. These findings suggest that STG supplementation promotes greater increases in the serum EPA concentration and EPA/AA ratio compared with PM, thereby supporting our initial hypothesis. However, the hypothesis regarding improvements in muscular endurance performance and reductions in muscle fatigue was not supported.

In this study, although the difference in the serum EPA concentration before and after 8 weeks of supplementation was not statistically significant between the STG and PM groups (p = 0.06), there was a trend toward a greater increase in the STG group (Figure 1A). The serum EPA/AA ratio was significantly higher in the STG group compared with the PM group (p < 0.05, Figure 1B). Our previous study demonstrated that 8 weeks of STG supplementation significantly increased serum EPA levels and the EPA/AA ratio compared with placebo [6]. However, no previous studies have compared STGs with PMs. To the best of our knowledge, this is the first study to demonstrate that STG supplementation results in a significantly greater increase in the EPA/AA ratio than PM. Increases in serum EPA and the EPA/AA ratio have been shown to contribute to the suppression of systemic inflammatory responses and improvements in energy metabolism through anti-inflammatory actions and enhanced lipid metabolism [26,27]. Moreover, they are widely recognized to play important roles in preventing the progression of arterial stiffness (atherosclerosis) and reducing the risk of cardiovascular disease [28,29]. Given that we observed a significant difference in the EPA/AA ratio between the STG and PM groups, STGs may have superior bioavailability than PMs. The structured triacylglycerols (STG) used in this study are designer lipids in which EPA/DHA and medium-chain fatty acids (MCFA) are re-esterified onto the same glycerol backbone and, upon ingestion, are digested and absorbed as a single triacylglycerol (TAG). By contrast, the physical mixture (PM) is a simple blend of an EPA/DHA-containing oil and an MCT oil without molecular coupling; during digestion the constituent oils are emulsified and hydrolyzed independently. Consequently, differences in TAG architecture can affect hydrolysis by pancreatic lipase, micelle formation, re-esterification, and chylomicron assembly, potentially yielding divergent effective uptake (bioavailability) of EPA/DHA, which are long-chain polyunsaturated fatty acids (LC-PUFA) [16]. In addition, whereas MCTs are rapidly digested and their MCFAs reach the liver via the portal vein and undergo prompt β-oxidation without requiring chylomicron formation, the co-localization of MCFA with EPA/DHA within a single TAG molecule in STG may alter physicochemical behavior in the digestive/emulsification phase (e.g. micellization and interfacial dynamics). This, in turn, could favor the absorption and systemic delivery of EPA/DHA (for example, elevating the EPA/AA ratio). Moreover, if MCFAs are co-absorbed with long-chain fatty acids as TAG and routed through the lymphatic system rather than being used primarily for hepatic energy production, they may be preferentially oxidized in peripheral tissues, thereby leaving EPA/DHA—as LC-PUFA—more available to function as bioactive mediators. This remains a working hypothesis.

Conversely, no significant differences were observed between the groups in the number of repetitions from the first to fourth set of the leg extensions or in the total number of repetitions (Figure 2A and B). In addition, there were no differences in blood lactate concentrations before and after exercise (Figure 2C). Furthermore, no significant correlations were found between the increases in the serum EPA concentration or the EPA/AA ratio and either the number of repetitions or lactate concentrations in either group. A previous study employing a similar leg extension task also failed to detect clear effects on repetition counts; however, a negative correlation was reported between increases in the serum EPA/AA ratio and the rate of decline in repetition times [6]. Based on this evidence, it is conceivable that increases in the EPA/AA ratio through STG supplementation may contribute to the attenuation of declines in muscular endurance. However, this relationship was not observed in the present study. One possible explanation is that the baseline EPA concentrations and EPA/AA ratios in our participants were already higher than those in previous research (e.g. Tsuchiya et al. [6] EPA = 19.5 vs. 12.1 μg/mL; EPA/AA ratio = 0.101 vs. 0.072) which may have limited the detectable additive effects of supplementation. Moreover, the relatively small sample size of this study (n = 28) may have increased susceptibility to inter-individual variability, thereby influencing the interpretation of the results. Furthermore, previous studies on the effects of EPA and DHA supplementation on endurance performance have yielded inconsistent findings [30,31], and no definitive conclusions can thus be drawn at present. Future research should employ larger-scale trials and study designs that account for participants' dietary and exercise habits to further investigate the relationship between the EPA/AA ratio and muscular endurance.

We also examined the effects of STG and PM supplementation on muscle fatigue following a muscular endurance exercise. The results showed no significant effects on any muscle fatigue markers (Table 2). Our previous study demonstrated that 8 weeks of STG supplementation significantly attenuated the post-exercise decline in MVC compared with placebo [6]. High-intensity muscle activity leads to an accumulation of metabolites within the muscle, which in turn increases the movement of serum components into muscle tissue through osmotic pressure [32]. This process results in reduced flexibility and muscle swelling. In addition, increased echo intensity has been considered to reflect higher water content within skeletal muscle [33]. Among various indicators of muscle fatigue, a reduction in MVC due to glycogen depletion is considered one of the most important measures for evaluating muscle fatigue [25,34–36]. The precise reason for the absence of group differences in the present study is unclear, but one possible explanation is that the workload of the leg extensions was relatively low. The low load may have led to a higher total number of repetitions and longer exercise duration, thereby increasing the contribution of mental fatigue. Indeed, the inter-individual variability in total repetitions was large (STG group: 165 ± 38.4 repetitions; PM group: 138.0 ± 87.3 repetitions), and post-exercise changes in muscle fatigue markers were relatively mild. Therefore, future studies should employ a high-intensity muscular endurance exercise model that imposes greater muscular demands to better compare the effects of STG and PM supplementation.

This study has several limitations. First, the supplementation period was limited to 8 weeks. With longer-term supplementation, it is possible that the increase in the serum EPA concentration in the STG group would have become significantly greater than that in the PM group, similar to the EPA/AA ratio, and that improvements in endurance performance and attenuation of muscle fatigue might also be observed. Thus, future research should examine the effects of prolonged supplementation. Second, the participants in this study were restricted to young men who did not engage in regular training. Different outcomes may be expected in athletes, women, or older adults. Therefore, further studies in broader populations, including athletes, women, and older adults, are warranted to examine whether STG oil intake can facilitate training adaptations. Finally, future studies should include a placebo group and use a three-arm design.

In conclusion, 8 weeks of supplementation with STGs containing EPA and DHA significantly increased the serum EPA/AA ratio compared with a PM, and no beneficial effects were observed in muscular endurance performance or muscle fatigue. By demonstrating differences in the bioavailability of EPA between STGs and PMs, this study suggests potential implications not only for sports performance but also for the prevention of lifestyle-related diseases, including cardiovascular disease. Future research should investigate the effects of longer-term supplementation and exercise protocols with different intensities.

Funding Statement

This study was supported by Nissui Ltd. However, the sponsor was not involved in data collection or data entry and there were no restrictions on publication.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

Please contact the author for data requests.

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee for Human Experiments at Teikyo Heisei University (ID: 2023-134) in compliance with the Declaration of Helsinki.

Consent for publication

Not applicable.