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This study aimed to compare 24-h and postprandial glucose responses to incremental intervals of standing (STAND), walking (WALK), and cycling (CYCLE) to a sit-only (SIT) condition.Nine overweight/obese (body mass index = 29 ± 3 kg·m−2) adults (30 ± 15 yr) participated in this randomized crossover full-factorial study, with each condition performed 1 wk apart. STAND, CYCLE, and WALK intervals increased from 10 to 30 min·h−1 (2.5 h total) during an 8-h workday. WALK (1.0 mph) and STAND were matched for upright time, and WALK and CYCLE were matched for energy expenditure (~2 METs). Continuous interstitial glucose monitoring was performed for 24 h to include the 8-h workday (LAB), after-work evening hours (EVE), and sleep (SLEEP). Three 2-h postprandial periods were also analyzed. Linear mixed models were used to test for condition differences.Compared with SIT (5.7 ± 1.0 mmol·L-1), mean 24-h glucose during STAND (5.4 ± 0.9 mmol·L−1) and WALK (5.3 ± 0.9 mmol·L−1) were lower, and CYCLE (5.1 ± 1.0 mmol·L−1) was lower than all other conditions (all P < 0.001). During LAB and EVE, mean glucose was lower for STAND, WALK, and CYCLE compared with SIT (P < 0.001). During SLEEP, the mean glucose for CYCLE was lower than all other conditions (P < 0.001). Compared with SIT, cumulative 6-h postprandial mean glucose was 5%–12% lower (P < 0.001) during STAND, WALK, and CYCLE, and 6-h postprandial glucose integrated area under the curve was 24% lower during WALK (P < 0.05) and 44% lower during CYCLE (P < 0.001).Replacing sitting with regular intervals of standing or light-intensity activity during an 8-h workday reduces 24-h and postprandial glucose. These effects persist during evening hours, with CYCLE having the largest and most sustained effect.