BACKGROUND: 2-Fucosyllactose (2-FL) is an oligosaccharide contained in human milk and possesses prebiotic and anti-inflammatory effects, which may alleviate skeletal muscle atrophy under caloric restriction. This study evaluated the impacts of 12 weeks of 2-FL supplementation in conjunction with exercise (10,000 steps/day, 5 days/week) and energy-reduced (-300 kcals/day) dietary interventions on changes in body composition and health-related biomarkers. METHODS: A total of 41 overweight and sedentary female and male participants (38.0 ± 13 years, 90.1 ± 15 kg, 31.6 ± 6.6 kg/m2, 36.9 ± 7% fat) took part in a randomized, double-blind, and placebo-controlled study. The participants underwent baseline assessments and were then assigned to ingest 3 g/day of a placebo (PLA) or Momstamin 2-F while initiating the exercise and weight-loss program. Follow-up tests were performed after 6 and 12 weeks. Data were analyzed using general linear model statistics with repeated measures and mean changes from baseline values with 95% confidence intervals (CIs). RESULTS: No group × time × sex interaction effects were observed, so group × time effects are reported. Participants in both groups saw comparable reductions in weight. However, those with 2-FL demonstrated a significantly greater reduction in the percentage of body fat and less loss of the fat-free mass. Additionally, there was evidence that 2-FL supplementation promoted more favorable changes in resting fat oxidation, peak aerobic capacity, IL-4, and platelet aggregation, with some minimal effects on the fermentation of short-chain fatty acids and monosaccharides in fecal samples. Moreover, participants perceptions regarding some aspects of the functional capacity and ratings of the quality of life were improved, and the supplementation protocol was well tolerated, although a small, but significant, decrease in BMC was observed. CONCLUSIONS: The results support contentions that dietary supplementation of 2-FL (3 g/d) can promote fat loss and improve exercise- and diet-related markers of health and fitness in overweight sedentary individuals initiating an exercise and weight-loss program. Further research is needed to explore the potential health benefits of 2-FL supplementation in both healthy and elderly individuals (Registered clinical trial #NCT06547801).