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We examined the effects of interrupting prolonged sitting with multiple 2-min walking breaks or one 30-min continuous walking session on glucose control and ambulatory blood pressure (ABP).Ten overweight/obese, physically inactive participants (five men; 32 ± 5 yr; BMI, 30.3 ± 4.6 kg·m−2) participated in this randomized four-trial crossover study, with each trial performed on a separate, simulated workday lasting 9 h: 1) 30 min of continuous moderate-intensity (30-min MOD) walking at 71% ± 4% HRmax; 2) 21 × 2 min bouts of moderate-intensity (2-min MOD) walking at 53% ± 5% HRmax, each performed every 20 min (42 min total); 3) 8 × 2 min bouts of vigorous-intensity (2-min VIG) walking at 79% ± 4% HRmax, each performed every hour (16 min total); 4) 9 h of prolonged sitting (SIT). Participants underwent continuous interstitial glucose monitoring and ABP monitoring during and after the simulated workday spent in the laboratory, with primary data analysis from 12:30 h to 07:00 h the next morning.Compared with SIT (5.6 ± 1.1 mmol·L−1), mean 18.7-h glucose was lower during the 2-min MOD (5.2 ± 1.1 mmol·L−1) and 2-min VIG (5.4 ± 0.9 mmol·L−1) trials and mean 18.7-h glucose during the 30-min MOD trial (5.1 ± 0.8 mmol·L−1) was lower than all other trials (P < 0.001). Postprandial glucose was approximately 7% to 13% lower during all trials compared with SIT (P < 0.001), with 30-min MOD having the greatest effect. Only the 30-min MOD trial was effective in reducing systolic ABP from 12:30 to 07:00 h (119 ± 15 mm Hg) when compared with SIT (122 ± 16 mm Hg; P < 0.05).Replacing sitting with 2-min MOD walking every 20 min or 2 min of vigorous-intensity walking every hour during a simulated workday reduced 18.7 h and postprandial glucose, but only 30-min MOD walking was effective for reducing both glucose and systolic ABP.