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Background: The effects of replacing time in specific activity categories for other categories (e.g. replacing sedentary time with light activity) on health and fitness are not well known. This study used isotemporal substitution to investigate the effects of substituting activity categories in an equal time exchange fashion on health and fitness in young people. Methods: Participants were drawn from schools in Camden, London (n = 353, mean age 9.3 ± 2.3 years). Time sedentary, in light and in moderate-to-vigorous activity (MVPA) was measured via accelerometry. The effects of substituting time in activity categories (sedentary, light and MVP A) with equivalent time in another category on health and fitness were examined using isotemporal substitution. Results: In single and partition models, MVPA was favourably associated with body fat %, horizontal jump distance and flexibility. Time sedentary and in light activity were not associated with health and fitness outcomes in these models. In substitution models, replacing one hour of sedentary time with MVPA was favourably associated with body fat % (B = -4.187; 95 % confidence interval (CI), -7.233, -1.142), horizontal jump distance (B =16.093; 95 % CI, 7.476, 24.710) and flexibility (B = 4.783; 95 % CI, 1.910, 7.656). Replacing time in light activity with MVPA induced similar benefits but there were null effects for replacing sedentary with light intensity. Conclusion: Substituting time sedentary and in light activity with MVPA was associated with favourable health and fitness. Time in sedentary behaviour may only be detrimental to health and fitness when it replaces time in MVPA in young people. [ABSTRACT FROM AUTHOR]