Abstract Objective Recent evidence suggests that short bouts of uninterrupted sedentary behavior reduce insulin sensitivity and glucose tolerance while increasing triglyceride levels in both healthy and overweight/obese adults. To date no study has examined the acute impact of uninterrupted sitting in children and youth. The objective of the present study was to determine whether 8h of uninterrupted sitting increases markers of cardiometabolic disease risk in healthy children and youth, in comparison to 8h of sitting interrupted by light intensity walk breaks or structured physical activity. Materials/Methods 11 healthy males and 8 healthy females between the ages of 10 and 14years experienced 3 conditions in random order: (1) 8h of uninterrupted sitting (Sedentary); (2) 8h of sitting interrupted with a 2-min light-intensity walk break every 20min (Breaks); and (3) 8h of sitting interrupted with a 2-min light-intensity walk break every 20min as well as 2×20min of moderate-intensity physical activity (Breaks+Physical Activity). Insulin, glucose, triglyceride, HDL and LDL cholesterol area under the curve were calculated for each condition. Results We observed no significant differences in the area under the curve for any marker of cardiometabolic disease risk across the 3 study conditions (all p>0.09). Conclusions These results suggest that in comparison to interrupted sitting or structured physical activity, a single bout of 8h of uninterrupted sitting does not result in measurable changes in circulating levels of insulin, glucose, or lipids in healthy children and youth.