Gender differences in metabolic response to lifestyle interventions remain poorly explored. This study aimed to evaluate the impact of a six-month Mediterranean diet (MD) intervention combined with regular physical activity on metabolic parameters in overweight adults. A prospective cohort study was conducted in an obesity clinic in Rome, Italy, involving overweight adults (BMI ≥ 25 kg/m2) motivated to improve their lifestyle. Participants (n = 205; 107 men and 98 women) self-selected into physical activity groups (aerobic, anaerobic, combined or no activity). Gender-specific metabolic changes were assessed, including lipid profiles, liver markers and fasting glucose. Significant gender differences in metabolic results were observed. Men showed greater reductions in total cholesterol (TC) and LDL, as well as significant reductions in alanine aminotransferase (ALT). Women showed a significant increase in HDL cholesterol. Fasting blood glucose decreased significantly in both sexes, with no differences between the sexes. Activity-specific analysis revealed that anaerobic activity significantly improved lipid metabolism in men, while aerobic activity produced the greatest benefits in women, including increased HDL and improved liver marker profiles. Therapeutic strategies combining MD and physical activity must take into account gender-specific physiological differences and the type of sport activity to optimise metabolic benefits. Personalised approaches may improve the management of cardiovascular risk factors in overweight individuals. This study is registered on ClinicalTrials.gov (NCT06661330).
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