BACKGROUND: The amount of time children spend in sedentary behaviours may have adverse health effects. OBJECTIVE: To examine the substitution effects of displacing a fixed duration of sedentary time with physical activity (PA) on children's body composition. METHODS: We included 386 children (197 boys). Outcomes were body mass index, waist circumference, total body fat mass and trunk fat mass assessed by dual-energy X-ray absorptiometry. Sedentary time and PA were measured with accelerometers. Data were analysed by isotemporal analyses estimating the effect of reallocating 15 and 30 min d-1 of sedentary time into light (light physical activity), and moderate-to-vigorous (MVPA) PA on body composition. RESULTS: Reallocating 15 and 30 min d-1 of sedentary time into MVPA was negatively associated with body fatness in cross-sectional analyses. Prospectively, reallocating 30 min of sedentary time into 30 min of MVPA was negatively associated with waist circumference (β = -1.11, p 0.05) with body fatness outcomes. CONCLUSIONS: Substituting sedentary time with MVPA using isotemporal analysis is associated with positive effects on body composition