Long-Term Omega-3 and Omega-3 Index >8%: Effects on Physiological Recovery

The proposed research will follow quantitative methods using a randomized independent-groups design. Participants will be allocated to one of two conditions: a placebo group or a fish oil supplementation group. All testing will take place in the St. Mary's University Performance Labs.

Screening and Eligibility

At the initial visit, participants will provide informed consent and complete a PAR-Q form. Eligibility screening will include confirmation that participants have an Omega-3 index ≤ 5%, assessed via a dried blood spot sample obtained from a capillary finger-prick. Samples are processed by OmegaQuant at their lab in Stirling, and results are typically returned within one week.

Participants will also be excluded if they are currently consuming ≥1 g/day of fish-oil supplements.

Randomisation and Blinding

Eligible participants will be randomly assigned to one of the two experimental groups using a random number generator (https://www.random.org). The study will follow a double-blind design: participants will not know which supplement they receive, and the primary investigators involved in data collection will also remain blinded. Supplement distribution and any dose adjustments will be conducted by a separate member of the research team who is unblinded and not involved in testing of participants or data analysis.

Screening Tests/familiarisation

Once Omega-3 index results are available, eligible participants will return to the lab for physiological screening/familiarisation of the test battery and to receive their supplements. Screening/familiarisation procedures include:

* An incremental treadmill ramp test, starting at 8 km·h-¹ and increasing by 1 km·h-¹ every 3 minutes until volitional exhaustion. During the test the below measures will be taken: * Capillary blood lactate sampling (finger or earlobe) every 3 minutes. * Continuous breath-by-breath respiratory analysis using the Vyntus online gas analyser. * Heart rate monitoring (Polar H10) and Rate of Perceived Exertion (RPE; Borg scale).

The screening session also introduces participants to the test battery illustrated in Figure 3.

Supplementation Phase (Part A)

Participants will be asked to refrain from:

* Alcohol for 48 hours before each testing session. * Caffeine for 12 hours before each testing session.

Participants will consume their assigned supplements daily for four months. Investigators will contact participants approximately every four weeks to assess adherence and to distribute additional supplement supplies.

At two months, a repeat dried blood spot sample will assess whether Omega-3 levels in the fish-oil group are increasing as expected. If required, supplementation will be adjusted using the OmegaQuant calculator (https://omegaquant.com/omega-3-index-calculator/). Adjustments will typically involve changes of approximately 0.5 g/day (EPA + DHA).

To maintain double-blinding, placebo dosages will also be altered by ±0.5 g/day, ensuring both investigator and participant remain unaware of group allocation.

After four months, participants will provide another dried blood sample. The aim is for the fish-oil group to reach an Omega-3 index ≥ 8%. Participants who do not meet their target will continue supplementation for an additional 2-4 weeks, followed by another blood test, until the required level is achieved. A cut-off point 6 additional weeks will be incorporated.

Completion of this phase constitutes Part A: the supplementation phase. Exercise-Induced Muscle Damage Trial (Part B) Approximately one week before Part B, participants will return to the lab to complete a second treadmill ramp test to determine the appropriate speed for the downhill running protocol.

Participants will then attend the laboratory on four consecutive days to complete pre- and post-exercise assessments associated with the exercise-induced muscle damage protocol.

The proposed study requires participants to take fish oil supplementation in order to increase their Omega-3 index and investigate whether this produces performance or recovery benefits, beyond the well-established long-term health effects associated with approximately 3 g/day of EPA + DHA. Fish oil supplementation is widely used in both clinical and general health contexts and is considered safe at doses of up to 5 g/day, as recognised by the European Food Safety Authority.

Participants will be recruited to complete the supplementation phase and take part in an exercise-induced muscle damage (EIMD) protocol. They will be randomly allocated to one of the two study groups (fish oil or placebo).

Fish oil supplementation will be provided by OmegaQuant (https://parasolnutrition.com/product/omega-3-fish-oil/) , this supplement is commercially available

Test battery:

1. Venous Blood Samples Venous blood samples will be collected to assess resting concentrations of inflammatory and muscle-damage biomarkers, including Interleukin-6 (IL-6), Interleukin-10 (IL-10), Tumor Necrosis Factor-α (TNF-α), myoglobin, lactate dehydrogenase (LDH), and creatine kinase (CK). Samples will be obtained by qualified phlebotomists at St Mary's University (Toby Helder, Prof. Charles Pedlar, and Dr Jessica Hill).

All venous samples will be stored at -80°C and later analysed using enzyme-linked immunosorbent assays (ELISAs). Venous sampling will occur at all required testing timepoints. 2. FORD/FORT Capillary Blood Testing

Fingertip capillary blood samples (50 µL and 20 µL, respectively) will be collected to assess oxidative stress using the Clini5 device (Callegari, Italy): * FORT: Free Oxygen Radical Test * FORD: Free Oxygen Radical Defence Test These provide an overall assessment of redox status. The device uses rapid, ready-prepared reagents requiring no cooling or manual preparation. Results are available within 6 minutes. 3. hs-CRP Capillary Blood Testing A 20 µL fingertip capillary sample will be collected to measure high-sensitivity C-reactive protein (hs-CRP), a marker of systemic inflammation. Analysis will be performed using the Eurolyser Cube S analyser (Eurolyser, Austria), with results available within 5 minutes. 4. Perceived Muscle Soreness

Participants will report muscle soreness using two validated scales: * The Visual Analogue Scale (VAS) * The Borg CR-10 Scale These will be administered at all relevant timepoints. 5. Maximal Strength Testing (Cybex Dynamometer)

Isokinetic Strength:

Peak torque of the knee extensors will be measured at 60°·s-¹ using a Cybex isokinetic dynamometer. This will be completed at all testing timepoints. 6. Isometric Mid-Thigh Pull (IMTP) Peak force and rate of force development will be assessed using an isometric mid-thigh pull rig with standardised knee and hip angles. Force production will be recorded via VALD force plates. 7. Countermovement Jump (CMJ) Participants will perform countermovement jumps on VALD force plates to assess neuromuscular function. They will complete three warm-up jumps, followed by three recorded trials, with hands placed on the hips to minimise upper-body contribution.

Downhill running protocol Participants will arrive in a fasted state either over night (for morning sessions) or a minimum of four hours fasted (mid-morning, afternoon and evening sessions). Participants will be familiarized with all equipment used and procedures before baseline measurements of height, weight and body composition are taken and pre-test venous blood sample taken. Participants will then complete a five-minute warm-up on a treadmill at 1% gradient at a self-selected speed. Following the warm-up, the treadmill will then be lowered to -10% gradient and participants will run for 45 minutes at 70% of their maximal aerobic capacity.

\- Participants will be required to take their assigned supplements daily for a period of four months. During this phase, they will have brief check-ins approximately every four weeks to monitor wellbeing and adherence. At the midpoint of supplementation (two months), a short visit is required for a dried blood spot test (see Figure 1 for an overview of the study timeline).

Although the overall study duration spans several months, data collection involves only nine in-person visits, most of which will last under one hour. Only the main exercise-testing sessions will be longer in duration.

* The research will take part at St Mary's University in the performance Laboratories. * All testing equipment used is the property of St Mary's University Twickenham. All supplement will be provided by OmegaQuant. All testing procedures are as outlined by British Association of Sport and Exercise Science.

• Muscle soreness (Visual Analogue Scale, VAS) [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]
• Muscle soreness (Borg CR-10 Scale) [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]
• Knee extensor strength (isokinetic dynamometry: peak torque at 60°/s) [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]
• Knee extensor isometric endurance (time to task failure at 50% peak torque) [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]
• Isometric mid-thigh pull (IMTP): peak force and rate of force development [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]
• Countermovement jump (CMJ): jump performance [Time frame: Participants will complete this once at baseline and then again after 4 months of supplementation.]