Obesity is a complex medical condition that is associated with a range of comorbidities, including hypertension, type 2 diabetes, dyslipidaemia, gastrointestinal disorders, joint pain, and musculoskeletal complications. Current treatment approaches for obesity primarily involve lifestyle modifications, including diet-induced weight loss and physical exercise. However, evidence from previous research highlights a concern regarding diet-induced body mass loss: approximately 25–30% of the total body mass lost is derived from fat-free mass (FFM). This decline in FFM is associated with diminished muscle mass and function, reduced metabolic rate, and an elevated risk of body mass regain. Attenuating FFM loss during body mass loss is therefore critical for healthy body mass loss. Long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFA) have been proposed as a potential strategy to mitigate these effects by influencing body composition, muscle mass and function, and inflammation during energy balance. Evidence suggests that LCn-3 PUFA can reduce fat mass while enhancing FFM, improving muscle mass, strength, and function, and mitigating inflammation. However, despite these potential benefits, the evidence supporting the efficacy of LCn-3 PUFA supplementation during diet-induced body mass loss on body composition, muscle function, and inflammatory markers remains limited and requires further exploration. The first aim of this thesis was to systematically investigate the effects of supplementation with LCn-3 PUFA during caloric restriction (CR) on body mass, fat mass and FFM loss (Chapter 2). Eleven studies were included in this systematic review and meta-analysis as they met the inclusion criteria of the systematic review, with a total of 637 participants. The participants’ age ranged between 18 and 61 years, with a mean BMI ranging between 27 and 36 kg/m2 . Pooled analyses showed that LCn-3 PUFA supplementation during CR had no additional effect on changes in body mass (SMD = -0.05: 95% CI -0.22 to 0.13; p = 0.62; I2 : 10%), BMI (SMD = -0.06, 95% CI -0.25 to 0.13; p = 0.55; I2 : 18%), fat mass (SMD = - 0.01; 95% CI -0.25 to 0.24; p = 0.96; I2 : 46%), or FFM (SMD = 0.12, 95%CI -0.14 to 0.37, p = 0.36; I2 :35%). The lack of impact of LCn-3 PUFA on body mass and composition observed in this systematic review (Chapter 2) may be attributed to some limitations in the iii included studies. Most of the studies assessed body composition using bioelectrical impedance analysis (BIA), applied low doses of LCn-3 PUFA, and also did not evaluate muscle strength during diet-induced body mass loss. To address the gaps identified in our systematic review, a double-blind, randomised, placebo-controlled trial (RCT) was conducted, including a 4-week preparation phase, an 8-week alternate-day fasting (ADF) phase, and an 8-week body mass maintenance phase, with participants taking 4 capsules/day of krill oil as a source of LCn-3 PUFA throughout (Chapter 4). Body composition was evaluated via the deuterium water (D2O) dilution method, and parameters of muscle function, and fasting blood samples were measured at the pre- and post body mass loss phase. Forty-one healthy adults completed this RCT. The two-way ANOVA revealed significant time and time*group interaction effects on FFM, handgrip strength, chair rising test, TNF-α, CRP, and systolic blood pressure (all p < 0.05). Post-intervention, there was a small, non-significant reduction in FFM (- 0.2 ± 0.9 kg, p > 0.05) and handgrip strength (-0.2 ± 0.5 kg, p > 0.05) in the krill oil group, whereas the placebo group experienced significant reductions in FFM (- 1.2 ± 2.0 kg, p < 0.05) and handgrip strength (-0.9 ± 0.7 kg, p < 0.05). The time to conduct the chair rising test decreased significantly in the krill oil group (-1.8 ± 0.9 s, p < 0.05), whereas the reduction in the placebo group was not significant (- 0.3 ± 1.2 s, p > 0.05). TNF-α levels decreased significantly in both groups (all p < 0.05), with a greater reduction in the krill oil group (-1.4 ± 0.2 pg/ml) compared to the placebo group (-0.9 ± 0.5 pg/ml). Similarly, CRP levels were significantly reduced in both groups (all p < 0.05), with a greater reduction in the krill oil group (-51.4 ± 25 ng/ml) than in the placebo group (-33.5 ± 12.6 ng/ml). Systolic blood pressure decreased significantly in both groups (all p < 0.05), with a greater reduction observed in the krill oil group (-9 ± 6 mmHg) compared to the placebo group (-4 ± 4 mmHg). No significant difference was observed in changes between groups in body mass, body fat, insulin, glucose HOMA-IR, TAG, or diastolic blood pressure (all p > 0.05). Therefore, from this RCT, it was concluded that supplementation with krill oil during diet-induced body mass loss via ADF helps to attenuate the associated decline of FFM and muscle function, improve functional capacity, and reduce TNF-α and CRP levels. Supplementation with LCn-3 PUFA, in the absence of CR, has been associated with appetite reduction and enhanced sensations of fullness and satiety in individuals iv living with overweight or obesity. However, the effects of LCn-3 PUFA supplementation during diet-induced body mass loss on appetite and gastrointestinal appetite hormones remain underexplored. In Chapter 5, the impact of LCn-3 PUFA during diet-induced body mass loss on changes in appetite and gastrointestinal appetite hormones was examined in a subset of the participants of the RCT (Chapter 4). This exploratory study included 28 adults (mean age: 39.4 ± 11.7 years; BMI: 27.9 ± 3.2 kg/m²) who participated in the RCT (Chapter 4). Body mass, body fat, and FFM were measured at baseline (week 4), at the end of the body mass loss phase (week 12), and at the end of the body mass maintenance phase (week 20). Fasting and postprandial subjective appetite scores, along with plasma concentrations of acylated ghrelin, Glucagon-Like Peptide-1 (GLP-1), and Peptide YY (PYY), were assessed before and after the body mass loss phase. The ANOVA revealed a significant time (p<0.05), but not group (p>0.05) or time*group interaction (p>0.05) effects for body mass, fat mass or FFM during the body mass loss phase. During the maintenance phase, no significant (p>0.05) time, group, or time*group interaction effects were found for body mass and FFM, but for fat mass, a significant time*group interaction effect was observed (p<0.05). During the maintenance phase, in the krill oil group, fat mass remained unchanged (p>0.05) but increased significantly (p< 0.05) in the placebo group. This coincided with the body mass loss-induced significant reduction (p<0.05) in the composite appetite score (CAS) in the krill oil but not the placebo group (p> 0.05). There was no significant (p>0.05) time, group, or time*group interaction effects for acylated ghrelin, GLP-1, and PYY during the body mass loss phase. Changes in body mass during the body mass loss and body mass maintenance phases were not correlated with acylated ghrelin, PYY, or GLP-1 (all p > 0.05). Body mass changes during the body mass loss phase showed a tendency toward a significant positive correlation with changes in CAS (r=0.36, p = 0.06). Therefore, krill oil supplementation during body mass maintenance may induce favourable changes in subjective appetite and prevent short-term fat mass regain. Overall, the current thesis demonstrates that supplementing with LCn-3 PUFA during diet-induced body mass loss is a promising strategy to attenuate the loss of FFM and muscle function. Beyond these benefits, LCn-3 PUFA supplementation also reduces inflammation and lowers blood pressure, underscoring its potential to enhance body composition, preserve muscle mass, and promote overall well- v being during body mass loss. Furthermore, LCn-3 PUFA supplementation may reduce subjective appetite and might help to prevent fat mass regain during the body mass maintenance phase, further supporting its role in long-term body mass management.

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