OBJECTIVES: Recent evidence suggests that occupational physical activity (OPA) is associated with adverse cardiovascular health, whereas leisure time physical activity is protective. This study explored explanatory physiological mechanisms. METHODS: Nineteen males (68% white, age=46.6+/-7.9 years, body mass index=27.9+/-5.1 kg/m(2)) with high self-reported OPA wore activity (ActiGraph and activPAL) and heart rate (HR) monitors for 7 days and an ambulatory blood pressure (BP) monitor on one workday and one non-workday. Mixed effects models compared cardiovascular variables (24-hour, nocturnal, waking and non-work time HR and BP) and nocturnal HR variability (HRV) on workdays versus non-workdays. Additional models examined associations of daily activity (steps, light physical activity (LPA) and moderate-to-vigorous physical activity (MVPA)) with cardiovascular variables. Workday by daily activity interactions were examined. RESULTS: 24-hour and waking HR and diastolic BP as well as non-work diastolic BP were significantly higher on workdays versus non-workdays (p<0.05 for all). However, no difference in systolic BP or nocturnal HR or BP was observed between work and non-workdays (p>0.05 for all). Low-frequency and high-frequency power indices of nocturnal HRV were lower on workdays (p<0.05 for both). Daily steps and LPA were positively associated with 24-hour and waking HR on work and non-workdays. Significant interactions suggested MVPA increases HR and lowers nocturnal HRV during workdays, with the opposite effect on non-workdays. CONCLUSIONS: Cardiovascular load was higher on workdays versus non-workdays with no compensatory hypotensive response following workdays. Daily MVPA may differentially affect ambulatory cardiovascular load and nocturnal HRV on workdays versus non-workdays, supporting the physical activity health paradox hypothesis.