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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.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).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 mM·h−1 (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.Alternating standing and sitting in 30-min bouts results in modest beneficial effects on postprandial glucose responses in overweight/obese office workers.