Interbeat interval data were collected from 219 Holstein cows in 2 smaller-scale farms and 3 larger-scale farms to investigate the effects of posture (standing vs. lying), rumination (rumination vs. no rumination) and feeding on baseline values of heart rate (HR) and heart rate variability (HRV) parameters reflecting vagal and sympathetic activity. A General Linear Model was used for detecting factors (parity, milking technology, herd size) having possible effects on HRV calculated for undisturbed lying posture. Basal values of cardiac parameters were also compared between larger and smaller-scale farms. Neither parity nor milking technology affected HRV parameters. Sympathetic activity increased in the order of lying, ruminating when lying, standing, ruminating when standing and feeding on both sizes of farms. Vagal activity decreased in that order in both lower- and larger-scale farms. Rumination caused an increase in HR and a decrease in vagal tone in both lower- and larger-scale farms and an increase in sympathetic activity during lying in both farm sizes. Basal vagal activity was lower in larger-scale farms compared to smaller-scale farms, while greater sympathetic activity was found in cows housed on larger-scale farms. Our findings demonstrate that reference values of HRV parameters in lactating dairy cows cannot be generally defined for Holstein cattle as they are affected by physical activity and herd size. Higher HR and sympathetic activity at rest in larger-scale farms compared to farms with lower cow population might be associated with higher levels of social stress and therefore should be considered as a potential welfare concern.