Excessive sitting and standing are proposed risk factors for cardiovascular diseases (CVDs), possibly due to autonomic imbalance. This study examines the association of objectively measured sitting and standing with nocturnal autonomic cardiac modulation. The cross-sectional study examined 490 blue-collar workers in three Danish occupational sectors. Sitting and standing during work and leisure were assessed during 1–5 days using accelerometers. Heart rate (HR) and heart rate variability (HRV) were obtained during nocturnal sleep as markers of resting autonomic modulation. The associations of sitting and standing still (h/day) with HR and HRV were assessed with linear regression models, adjusted for age, gender, body mass index, smoking, and physical activity. More sitting time during leisure was associated with elevated HR (p = 0.02), and showed a trend towards reduced HRV. More standing time at work was associated with lower HR (p = 0.02), and with increased parasympathetic indices of HRV (root mean squared successive differences of R-R intervals p = 0.05; high-frequency power p = 0.07). These findings, while cross-sectional and restricted to blue-collar workers, suggest that sitting at leisure is detrimental to autonomic cardiac modulation, but standing at work is beneficial. However, the small effect size is likely insufficient to mitigate the previously shown detrimental effects of prolonged standing on CVD.