The primary objective was to test the hypothesis that increased frequency of interruptions in prolonged sitting reduces postprandial glycemia independent of energy intake and expenditure.Healthy, sedentary, centrally obese men (n = 14; age*, 28.2 (23.4; 38.3) years; BMI, 31.9 ± 6.7 kg/m; VOmax*, 39.5 (38.8; 40.9) ml/min/kg; HbA1c, 5.3 ± 0.4% (34.1 ± 4.2 mmol/mol); mean ± SD (*median (25th; 75th percentile)) completed four 8-h interventions in randomized order: 1) uninterrupted sitting (SIT), 2) sitting interrupted by 2 min of walking (~30% of VO) every 20th minute (INT20), 3) sitting interrupted by 6 min of walking every hour (INT60), and 4) sitting interrupted by 12 min of walking every second hour (INT120). A standardized test drink was served at the beginning of and 4 h into the intervention (total of 2310 ± 247 kcal; 50% energy from carbohydrate, 50% energy from fat). Outcomes included the difference in the 8-h total area under the curve (tAUC) for primarily plasma glucose, and secondarily plasma insulin and C-peptide during INT20, INT60, and INT120 compared to SIT.2No difference [95% CI] was observed in the primary outcome, the 8-h tAUC for the plasma glucose, during INT20, INT60, and INT120 compared to SIT (-65.3 mmol/l∗min [-256.3; 125.7], +53.8 mmol/l∗min [-143.1; 250.8], and +18.6 mmol/l∗min [-172.4; 209.6], respectively).Interrupting sitting with increasing frequency did not reduce the postprandial plasma glucose response to prolonged sitting in healthy, sedentary, centrally obese men.