Objectives: We investigated associations between daily-life physical activity (PA) and prospective weight gain in non-obese people. We also examined whether these associations were independent of other lifestyle factors and changes in muscle mass and whether they were age-dependent and changed over a person’s life course. Methods: The data were extracted from the Lifelines cohort study (N=52,498; 43.5% men) and excluded obese individuals (BMI>30kg/m 2 ). We used the validated SQUASH questionnaire to estimate moderate-to-vigorous (MVPA; MET≥4), moderate (MPA; MET between 4 and 6.5) and vigorous PA (VPA; MET≥6.5) within non-occupational (commuting and leisure) and occupational domains. Body weight was objectively measured and changes were standardized to a 4-year period. Separate analyses, adjusted for age, educational level, diet, smoking, alcohol consumption and changes in creatinine excretion (a marker of muscle mass), were performed for men and women. Results: The average weight gain was +0.45±0.03 kg in women. Relative to each reference groups (No-MVPA, No-MPA and No-VPA), non-occupational MVPA (Beta (95%CI): -0.34 kg (-0.56;-0.13)), MPA (-0.32 kg (-0.54;-0.10)) and VPA (-0.30 kg (-0.43;-0.18)) were associated with less gain in body weight in women after adjusting for potential confounders, described above. These associations were dose-dependent when physically active individuals were divided in tertiles. Beta-coefficients (95%CI) for the lowest, middle, and highest MVPA tertiles relative to the ‘No-MVPA’ were, respectively, -0.24 (-0.47;-0.02), -0.31 (-0.53;-0.08), and -0.38 (-0.61;-0.16) kg. The average weight gain in men was +0.13±0.03 kg, and only non-occupational VPA was associated with less body weight gain. Beta-coefficients (95%CI) for the VPA tertiles relative to the ‘No-VPA’ group were, respectively, -0.25 (-0.42;-0.09), -0.19 (-0.38;-0.01) and -0.20 (-0.38;-0.02) kg. However, after adjusting for potential confounders, the association was no longer significant in men. The potential benefits of non-occupational PA were age-stratified and mainly observed in younger adults (men: <35 years; women: <55 years). Moreover, occupational MVPA was not associated with favourable changes in body weight in males and females. Conclusion: Higher non-occupational MVPA, MPA, and VPA were associated with less weight gain in women <55 years. In younger men (<35 years), only non-occupational VPA was associated with less weight gain.