The behavioural impact of an imposed bout of prolonged sitting is yet to be investigated in the paediatric population. The objective of the present study was to determine the acute effect of prolonged sitting on ad libitum food intake and spontaneous physical activity (PA) levels in healthy children and youth. A total of twenty healthy youth (twelve males and eight females) aged 10-14 years, with a mean BMI of 18·6 (sd 4·3) kg/m², were exposed to three experimental conditions in a random order: (1) a day of uninterrupted sitting (Sedentary); (2) a day of sitting interrupted with a 2 min light-intensity walk break every 20 min (Breaks); (3) a day of sitting interrupted with a 2 min light-intensity walk break every 20 min as well as 2 × 20 min of moderate-intensity PA (Breaks+PA). Food intake (ad libitum buffet meal) and PA (accelerometry for 24 h) were assessed following exposure to each experimental condition. Despite significant differences in sedentary behaviour and activity levels during the three in-laboratory sessions (all P< 0·01), we did not observe any differences in ad libitum food intake immediately following exposure to each experimental condition or any changes in the levels of sedentary behaviour or PA in the 24 h following exposure to each experimental condition (all P>0·25). These findings suggest that children and youth may not compensate for an imposed bout of sedentary behaviour by reducing subsequent food intake or increasing PA levels.