Background Twenty-four-hour movement behaviours are gaining attention in the research community. However, no study has addressed how 24-h activity profiles vary between structured and less structured days and whether an unfavourable activity profile is associated with childhood obesity. We aimed to analyse differences between school day and weekend day 24-h activity profiles and their associations with adiposity indicators among children and adolescents. Methods Participants were 382 children and 338 adolescents who wore wrist accelerometers for 24 hours a day for seven consecutive days. The 24-h activity profile expressed by the average acceleration (AvAcc) and intensity gradient (IG) were estimated from multi-day raw accelerometer data. Adiposity indicators included body mass index (BMI) z-score, fat mass percentage (FM%), fat mass index (FMI), and visceral adipose tissue (VAT). Multiple linear regression of activity profile metrics and adiposity indicators was performed separately for school and weekend days. Results Weekend days AvAcc and IG were lower compared to school days in both age groups (p <0.001 for all). Specifically, AvAcc was lower by 9.4% and 11.3% in children and adolescents, respectively. IG on weekend days was lower (more negative) by 3.4% in children and 3.1% in adolescents. Among children, on school days AvAcc and IG were negatively associated with FM%, FMI, and VAT, whilst on weekend days AvAcc was positively associated with BMI z-score, FMI, and VAT (p < 0.05 for all). Among adolescents, negative associations were found between weekend day AvAcc and IG and FM% and FMI (p < 0.05 for all), respectively. Conclusions This study confirms the importance of 24-h activity profile as a potentially protective factor against excess adiposity. The variability of movement behaviours during structured and less structured days should be considered when optimizing the 24-h movement behaviours to prevent childhood obesity.