Current physical activity (PA) guidelines recommend reducing prolonged sitting in addition to increasing PA. In this regard, the complementary effects of standing breaks to interrupt sitting, in addition to exercise, have not yet been evaluated. This study tested whether exercise alone or exercise combined with standing breaks enhances cardiometabolic outcomes compared with prolonged sitting in sedentary adults with dyslipidaemia. In this randomized cross-over trial, sixteen adults (mean ± SD: age = 47.8 ± 9.3 years; BMI = 24.6 ± 2.7 kg/m2; 8 women) completed three two-day conditions: prolonged sitting (SIT), prolonged sitting + exercise (FIT), and sitting interrupted with standing breaks (30 min/h) + exercise (MIX). The protocol was conducted in free-living, desk-based work environments, with a single 60-min moderate-intensity cycle ergometer bout performed on the second day. PA and sedentary behavior (SB) were objectively measured and standardized test meals were provided during the laboratory-based metabolic testing day. Postprandial metabolism (primary outcome) including glucose, insulin and triglycerides, was measured over 6 h. Linear mixed effects models with correction for baseline values were used to evaluate condition effects. Postprandial triglyceride (p = 0.112) and glucose (p = 0.150) concentrations did not differ across conditions whereas postprandial insulin responses were significantly lower in MIX than SIT (p = 0.041) and FIT (p = 0.010). Standing breaks combined with exercise did not reduce postprandial triglyceride or glucose concentrations, but improved insulin responses more than exercise alone in sedentary adults with dyslipidaemia. These findings suggest that interrupting prolonged sitting may provide additional metabolic benefits alongside structured exercise in at-risk populations. Trial Registration: https://clinicaltrials.gov/study/NCT06277713 (Registered February 12, 2024).