PURPOSE: This study aimed to determine whether interrupting prolonged sitting with three different walking-bout schedules improves glycemic metabolism relative to continuous prolonged sitting in sedentary adults. METHODS: In a randomized, crossover trial, 16 inactive healthy adults (7 men, 24 ± 3 yr old) completed four 26-h laboratory sessions, including 22.5 h in an energy expenditure (EE)-testing calorimeter chamber. The four 9-h intervention periods were as follows: 9-h uninterrupted sitting (SIT), 30-min sitting/3-min brisk (60% V˙O2max) treadmill walk (WALK3), 45-min sitting/5-min (WALK5), or 60-min sitting/8-min (WALK8). Coprimary outcomes included the difference in the mean interstitial glucose concentration, total area under the curve (tAUC), and incremental area under the curve (iAUC) for the entire 26 and 9 h (intervention period) during three activity-bout conditions compared with SIT. RESULTS: Compared with SIT, the 26-h mean glucose concentration was attenuated in WALK8 (Δmean -0.22 mmol·L [95% confidence interval = -0.43 to -0.001], P = 0.048) without adjustment for EE and attenuated in WALK3 (Δ-0.47 mmol·L [-0.75 to -0.10]), WALK5 (Δ-0.47 mmol·L [-0.83 to -0.10]), and WALK8 (Δ-0.53 mmol·L [-0.92 to -0.13]) after adjustment for EE (all P < 0.01). The 26-h tAUC was reduced in WALK3 (Δ-11.18 mmol·L per 26 h [-20.07 to -2.29]), WALK5 (2.12.67 mmol·L per 26 h [-22.54 to -2.79]), and WALK8 (Δ-13.85 mmol·L per 26 h [-24.60 to -3.10]) (all P < 0.01), as well as the iAUC (all P < 0.05), only after adjustment for EE. The 9-h mean glucose concentration, tAUC, and iAUC decreased in the three activity-break conditions regardless of EE adjustment (all P < 0.05). CONCLUSIONS: All three walking-bout conditions improved glycemic metabolism compared with SIT, independent of EE, in inactive, healthy adults.