Heart rate variability (HRV), as a physiological measure of animal welfare, was investigated in 36 cows milked in a parallel milking parlor with nonvoluntary exit. Heart rate variability parameters measured during the morning resting (baseline period) were compared with those measured during different stages of the entire milking process. No differences were found in HRV parameters between the baseline period, preparation, and main milking. A considerable reduction in vagal activity was detected during the movement of the cows to the milking parlor (driving) and while cows were in the holding area. The parasympathetic measures of HRV decreased whereas the sympatho-vagal balance increased compared with baseline. The same pattern was observed regarding the stage between removing the teat cups and leaving the milking parlor (waiting). No differences in any sympathetic measures were observed between the baseline period and any of the milking stages. These findings indicate that the milking process itself (preparation and main milking) is not stressful for cows. Decreased parasympathetic activity during driving might be the result of the physical activity of the cows, whereas waiting in the holding area and in the milking stall after milking caused stress for animals.