Dairy cows are frequently separated from their calves immediately after birth. However, recent welfare concerns from the public have brought this common practice into question. There is a lack of research observing the effects of delaying cow-calf separation. Thus, the objective was to delay the separation of cow-calf pairs and subsequently observe the behavior of the cows after the removal of the calf. Holstein dairy cows were allowed to maintain contact with their newborn calf for 0 (0D; n=6), 3 (3D; n=5), 5 (5D; n=6), or 7 days (7D; n=6) to evaluate the effect on cow behavior after delaying separation. After parturition, cow-calf pairs were moved to experimental pens for observation. At enrollment, video cameras were set up to continuously record the pairs, and activity monitors were attached to the cow’s leg. Heart rate monitors were placed on each cow for 4 hours after morning milking on the day before and the day of separation. All cows who had contact with their calves did not reject them, spent time grooming them and there were no injuries observed. Cows in the 5D and 7D treatment groups had a stronger behavioral response to separation than those in the 0D and 3D groups as seen in the increased number of vocalizations, greater heart rate standard deviations, and decreased lying time.