BACKGROUND: Uninterrupted prolonged sitting and exaggerated psychobiological reactivity to acute psychological stress are associated with increased risk of cardiovascular disease (CVD). Breaking up prolonged sitting with frequent, short bouts of light-intensity physical activity acutely lowers CVD risk markers under resting conditions. PURPOSE: To examine whether frequent interruptions to prolonged sitting with body-weighted resistance activity can acutely lower systolic blood pressure (SBP; primary outcome) and other cardiovascular (CV), inflammatory, and cortisol (secondary outcomes) responses to acute psychological stress. METHODS: This randomized crossover trial included 17 sedentary participants (9 men; mean ± SD age; 24.0 ± 0.5 years) who completed two conditions: (i) interrupting 4 hr of sitting with 4 min of light body-weighted resistance activity every 30-min (BREAK), and (ii) 4 hr of uninterrupted sitting (SIT). Following the BREAK and SIT intervention windows, CV, inflammatory, and cortisol markers were measured at rest, during stress tasks (8-min Paced Auditory Serial Addition Test [PASAT] and 3-min cold pressor [CP]), and 45-min recovery periods. RESULTS: There were main effects of time for CV parameters (SBP, diastolic blood pressure, heart rate, cardiac output, and total peripheral resistance [all p < .001]), inflammatory markers (interleukin-6 [IL-6]), and cortisol (p < .05) in response to stress. Time-by-condition interaction effects revealed that in the BREAK-condition there was lower SBP during immediate recovery from the CP (mean {95% confidence interval [CI]}: 127.2 [121.3, 133.4] vs 133.4 [125.5, 141.7] mmHg; p = .020), higher concentrations of plasma IL-6 45-min post-PASAT (2.70 [1.97, 3.70] vs 1.71 [1.32, 2.22] pg/mL; p = .010), and larger (nonsignificant) salivary cortisol concentrations 8-min post-CP (6.29 [4.60, 8.58] vs 3.97 [3.16, 4.99] nmol/L; p = .079). CONCLUSIONS: Interrupting prolonged sitting with frequent bouts of light intensity body-weighted resistance activity alters psychobiological responses to acute psychological stress. Further research should explore the longer-term implications for CVD risk.