Prolonged sitting is associated with increased risk for cardiovascular diseases and mortality. However, research into the physiological determinants underlying this relationship is still in its infancy. The aim of the study was to determine the extent to which occupational and leisure-time sitting are associated with nocturnal heart rate variability (HRV) in blue-collar workers. The study included 138 blue-collar workers (mean age 45.5 (SD 9.4) years). Sitting-time was measured objectively for four days using tri-axial accelerometers (Actigraph GT3X+) worn on the thigh and trunk. During the same period, a heart rate monitor (Actiheart) was used to sample R-R intervals from the electrocardiogram. Time and frequency domain indices of HRV were only derived during nighttime sleep, and used as markers of cardiac autonomic modulation. Regression analyses with multiple adjustments (age, gender, body mass index, smoking, job-seniority, physical work-load, influence at work, and moderate-to-vigorous physical activity) were used to investigate the association between sitting time and nocturnal HRV. We found that occupational sitting-time was negatively associated (p < 0.05) with time and frequency domain HRV indices. Sitting-time explained up to 6% of the variance in HRV, independent of the covariates. Leisure-time sitting was not significantly associated with any HRV indices (p > 0.05). In conclusion, objectively measured occupational sitting-time was associated with reduced nocturnal HRV in blue-collar workers. This indicates an attenuated cardiac autonomic regulation with increasing sitting-time at work regardless of moderate-to-vigorous physical activity. The implications of this association for cardiovascular disease risk warrant further investigation via long-term prospective studies and intervention studies.