BACKGROUND: Compared with prolonged sitting, regular activity breaks immediately lower postprandial glucose and insulin, but not triglyceride responses. Postprandial triglycerides can be lowered by physical activity but the effect is often delayed by ∼12 to 24 hours. OBJECTIVE: The objective of the study was to determine whether regular activity breaks affect postprandial triglyceride response in a delayed manner similar to physical activity. METHODS: In a randomized crossover trial, 36 adults (body mass index 23.9 kg/m [standard deviation 3.9]) completed four 2-day interventions: (1) prolonged sitting (SIT); (2) prolonged sitting with 30 minutes of continuous walking (60% VO), at the end of Day 1 (SIT + PA); (3) Sitting with 2 minutes of walking (60% VO) every 30 minutes (RAB); (4) A combination of the continuous walking and regular activity breaks in 2 and 3 above (RAB + PA). Postprandial plasma triglyceride, nonesterified fatty acids, glucose, and insulin responses were measured in venous blood over 5 hours on Day 2. RESULTS: Compared with SIT, both RAB (difference: -43.61 mg/dL·5 hours; 95% confidence interval [CI] -83.66 to -2.67; P = .035) and RAB + PA (-65.86 mg/dL·5 hours; 95% CI -112.14 to -19.58; P = .005) attenuated triglyceride total area under the curve (tAUC). RAB + PA produced the greatest reductions in insulin tAUC (-23%; 95% CI -12% to -31%; P < .001), whereas RAB resulted in the largest increase in nonesterified fatty acids (tAUC, 10.08 mg/dL·5 hours; 95% CI 5.60-14.84; P < .001). There was no effect on glucose tAUC (P = .290). CONCLUSIONS: Postprandial triglyceride response is attenuated by regular activity breaks, when measured ∼24 hours after breaks begin. Combining regular activity breaks with 30 minutes of continuous walking further improves insulinemic and lipidemic responses.