BackgroundWhile there is evidence that physical activity, sedentary behaviour (SB) and sleep may all be associated with modified levels of inflammatory markers in adolescents and children, associations with one movement behaviour have not always been adjusted for other movement behaviours, and few studies have considered all movement behaviours in the 24-hour day as an exposure.PurposeThe aim of the study was to explore how longitudinal reallocations of time between moderate-to-vigorous physical activity (MVPA), light physical activity (LPA), SB and sleep are associated with changes in inflammatory markers in children and adolescents.MethodsA total of 296 children/adolescents participated in a prospective cohort study with a 3-year follow-up. MVPA, LPA and SB were assessed by accelerometers. Sleep duration was assessed using the Health Behavior in School-aged Children questionnaire. Longitudinal compositional regression models were used to explore how reallocations of time between movement behaviours are associated with changes in inflammatory markers.ResultsReallocations of time from SB to sleep were associated with increases in C3 levels (difference for 60 min/d reallocation [d60] = 5.29 mg/dl; 95% confidence interval [CI] = 0.28, 10.29) and TNF-α (d60= 1.81 mg/dl; 95% CI = 0.79, 15.41) levels. Reallocations from LPA to sleep were also associated with increases in C3 levels (d60= 8.10 mg/dl; 95% CI = 0.79, 15.41). Reallocations from LPA to any of the remaining time-use components were associated with increases in C4 levels (d60ranging from 2.54 to 3.63 mg/dl;p< 0.05), while any reallocation of time away from MVPA was associated with unfavourable changes in leptin (d60ranging from 3088.44 to 3448.07 pg/ml;p< 0.05).ConclusionsReallocations of time between 24-h movement behaviours are prospectively associated with some inflammatory markers. Reallocating time away from LPA appears to be most consistently unfavourably associated with inflammatory markers. Given that higher levels of inflammation during childhood and adolescence are associated with an increased risk of chronic diseases in adulthood, children and adolescents should be encouraged to maintain or increase the level of LPA to preserve a healthy immune system.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12966-023-01471-9.