Purpose: This study aimed to examine whether reductions in sitting time through alternating 30-min bouts of sitting and standing can reduce postprandial glucose, insulin, and triglyceride responses. Methods: Twenty-three overweight/obese sedentary office workers (17 males and six females; mean ? SD: age, 48.2 ? 7.9 yr; body mass index, 29.6 ? 4.0 kg?m-2) undertook two short-term (5 d) experimental conditions in an equal, randomized (1:1) order. In a simulated office environment, participants performed typical occupational tasks for 8 h?d-1 while in a 1) seated work posture (control condition) or 2) interchanging between a seated and standing work posture every 30 min using an electric, height-adjustable workstation (intervention condition). Fasting and postprandial blood samples after a mixed test drink were collected hourly for 4 h on days 1 and 5 of each condition to assess serum insulin, plasma glucose, and triglycerides. Dietary intake (kJ?d-1) and physical activity were standardized during each condition. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12611000632998). Results: After adjustment for time (days 1 and 5), incremental area under the analyte time curve differed significantly between conditions for plasma glucose (P = 0.007) but not for serum insulin or plasma triglycerides. Adjusted mean glucose incremental area under the analyte time curve was lowered by 11.1 after the intervention condition (6.38 mMIhj1 (confidence interval, 5.04-7.71)) relative to the control condition (7.18 mM?h-1 (confidence interval, 5.85-8.52)). No temporal changes (days 1 vs 5) between conditions were observed. Conclusions: Alternating standing and sitting in 30-min bouts results in modest beneficial effects on postprandial glucose responses in overweight/obese office workers.