OBJECTIVE: To determine whether breaking up prolonged sitting with short bouts of standing or walking improves postprandial markers of cardiometabolic health in women at high risk of type 2 diabetes. RESEARCH DESIGN AND METHODS: Twenty-two overweight/obese, dysglycemic, postmenopausal women (mean ± SD age 66.6 ± 4.7 years) each participated in two of the following treatments: prolonged, unbroken sitting (7.5 h) or prolonged sitting broken up with either standing or walking at a self-perceived light intensity (for 5 min every 30 min). Both allocation and treatment order were randomized. The incremental area under the curves (iAUCs) for glucose, insulin, nonesterified fatty acids (NEFA), and triglycerides were calculated for each treatment condition (mean ± SEM). The following day, all participants underwent the 7.5-h sitting protocol. RESULTS: Compared with a prolonged bout of sitting (iAUC 5.3 ± 0.8 mmol/L ⋅ h), both standing (3.5 ± 0.8 mmol/L ⋅ h) and walking (3.8 ± 0.7 mmol/L ⋅ h) significantly reduced the glucose iAUC (both P < 0.05). When compared with prolonged sitting (548.2 ± 71.8 mU/L ⋅ h), insulin was also reduced for both activity conditions (standing, 437.2 ± 73.5 mU/L ⋅ h; walking, 347.9 ± 78.7 mU/L ⋅ h; both P < 0.05). Both standing (-1.0 ± 0.2 mmol/L ⋅ h) and walking (-0.8 ± 0.2 mmol/L ⋅ h) attenuated the suppression of NEFA compared with prolonged sitting (-1.5 ± 0.2 mmol/L ⋅ h) (both P < 0.05). There was no significant effect on triglyceride iAUC. The effects on glucose (standing and walking) and insulin (walking only) persisted into the following day. CONCLUSIONS: Breaking up prolonged sitting with 5-min bouts of standing or walking at a self-perceived light intensity reduced postprandial glucose, insulin, and NEFA responses in women at high risk of type 2 diabetes. This simple, behavioral approach could inform future public health interventions aimed at improving the metabolic profile of postmenopausal, dysglycemic women.