Objectives: Standing desks and stability balls are increasingly popular to increase muscle activity and thereby prevent potential adverse cardiometabolic effects of prolonged sitting. The present study examined the effects of (1) sitting on a stability ball (‘active sitting’) and (2) hourly 10-min standing interruptions during prolonged sitting on postprandial cardiometabolic biomarkers. Design: Experimental crossover study. Methods: Twenty healthy-weight males (19.2 ± 0.6 years) participated randomly in three 5-h conditions: (1) sitting on an office chair (SIT), (2) sitting on a stability ball (SIT-ACTIVE) and (3) sitting with hourly 10-min standing interruptions (SIT-STAND). In each condition, participants consumed a standardized mixed meal at baseline. Hourly blood samples and pre/post saliva samples were collected and analyzed for levels of insulin, glucose and cortisol. Pre/post hemodynamic monitoring (middle finger; Nexfin-monitoring) was conducted; heart rate was measured continuously (Polar) and muscle activity (leg and lower-back, Portilab) was measured during periods of sitting (on an office chair and on a stability ball) and standing. Results: Muscle activity and heart rate during standing periods were significantly higher than during sitting (both SIT and SIT-ACTIVE). Generalized estimating equations revealed no significant difference in any of the biomarkers between the three experimental conditions. Systolic blood pressure was lower during SIT-STAND, while stroke volume was lower during SIT-ACTIVE than during SIT. Although significant, these differences were small, approximating the day-to-day variability in blood pressure and stroke volume. Conclusions: We conclude that hourly standing interruptions during 5 h prolonged sitting or continuously sitting on a stability ball do not significantly affect postprandial cardiometabolic biomarkers in healthy young men. Trial registration: This trial is registered in the NTR trial register (NTRcode 5723).