Abstract Background Postprandial glucose, insulin, and triglyceride metabolism is impaired by prolonged sitting, but enhanced by exercise. The aim of this study was to assess the effects of a continuous exercise bout with and without intermittent active interruptions to prolonged sitting on postprandial glucose, insulin, and triglycerides. Methods Sedentary adults who were overweight to obese (n = 67; mean age 67 yr SD ± 7; BMI 31.2 kg∙m^{− 2} SD ± 4.1), completed three conditions: SIT: uninterrupted sitting (8-h, control); EX+SIT: sitting (1-h), moderate-intensity walking (30-min), uninterrupted sitting (6.5-h); EX+BR: sitting (1-h), moderate-intensity walking (30- min), sitting interrupted every 30-min with 3-min of light-intensity walking (6.5 h). Participants consumed standardized breakfast and lunch meals and blood was sampled at 13 time-points. Results When compared to SIT, EX+SIT increased total area under the curve (tAUC) for glucose by 2% [0.1–4.1%] and EX+BR by 3% [0.6–4.7%] (all p < 0.05). Compared to SIT, EX+SIT reduced insulin and insulin:glucose ratio tAUC by 18% [11–22%] and 21% [8–33%], respectively; and EX+BR reduced values by 25% [19–31%] and 28% [15–38%], respectively (all p < 0.001 vs SIT, all p < 0.05 EX+SIT-vs-EX+BR). Compared to SIT, EX+BR reduced triglyceride tAUC by 6% [1–10%] (p = 0.01 vs SIT), and compared to EX+SIT, EX+BR reduced this value by 5% [0.1–8.8%] (p = 0.047 vs EX+SIT). The magnitude of reduction in insulin tAUC from SIT-to-EX+BR was greater in those with increased basal insulin resistance. No reduction in triglyceride tAUC from SIT-to-EX+BR was apparent in those with high fasting triglycerides. Conclusions Additional reductions in postprandial insulin-glucose dynamics and triglycerides may be achieved by combining exercise with breaks in sitting. Relative to uninterrupted sitting, this strategy may reduce postprandial insulin more in those with high basal insulin resistance, but those with high fasting triglycerides may be resistant to such intervention-induced reductions in triglycerides. Trial registration Australia New Zealand Clinical Trials Registry (ACTRN12614000737639).