Background: Overweight women are at increased risk of metabolic dysfunction, muscle loss, and reduced physical function during middle age. Resistance training (RT), combined with a high-protein diet and omega-3 supplementation, may help mitigate these risks; however, their combined effects remain unclear. Objective: To examine whether omega-3 supplementation enhances the effects of RT combined with a high-protein diet on body composition, muscular performance, and selected biochemical markers in overweight women. Methods: Fifty-four overweight women (40–53 years) were randomly assigned to RT plus omega-3 supplementation with a high-protein diet (RO), RT plus placebo with a high-protein diet (RP), or a non-training control group (C). The RT intervention was performed three times per week for 8 weeks. Body composition, muscular performance, and circulating markers related to muscle metabolism and clinical safety were assessed before and after the intervention. Results: Forty-four participants completed the study. Both intervention groups demonstrated significant reductions in body mass and fat mass, alongside increases in skeletal muscle mass (SMM) and improvements in muscular strength, endurance, and power compared with the C group (p < 0.001). Markers related to muscle metabolism improved in both RT groups, with greater changes observed in the RO group. Clinical safety markers remained within normal ranges, with no between-group differences. Conclusions: Eight weeks of RT combined with a high-protein diet effectively improved body composition, muscle function, and anabolic signaling in overweight women. Short-term omega-3 supplementation selectively modulated biochemical markers but did not provide additional improvements in SMM, performance, or clinical safety markers, suggesting that its benefits may be limited without longer-term or higher-dose interventions.

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