The aim was to compare the behavioral response to full separation of cows and calves maintained together for 100 days or 24 h. Twelve Holstein-Friesian cow-calf pairs were enrolled into either treatment or industry groups (n = 6 cow-calf pairs/group). The treatment cows and calves were maintained on pasture together for 106±8.6 d and temporarily separated twice a day for milking. The Industry cows and their calves, were separated within 24 h postpartum. Triaxial accelerometer neck-mounted sensors were fitted to cows 3 weeks before separation to measure hourly rumination and activity. Before separation, cow and calf behavior was observed by scan sampling for 15 min. During the separation process, frequency of vocalizations and turn arounds were recorded. At separation, cows were moved to an observation pen where behavior was recorded for 3 d. A CCTV camera was used to record video footage of cows within the observation pens and behavior was documented from the videos in 15 min intervals across the 3 d. Before separation, industry calves were more likely to be near their mother than Treatment calves. During the separation process, vocalization and turn around behavior was similar between groups. After full separation, treatment cows vocalized three times more than industry cows. However, the frequency of time spent close to barrier, standing, lying, walking, and eating were similar between industry and treatment cows. Treatment cows had greater rumination duration, and were more active, than industry cows. These findings suggest a similar behavioral response to full calf separation and greater occurrence of vocalizations, from cows maintained in a long-term, pasture-based, cow-calf rearing system when compared to cows separated within 24 h. However, further work is required to assess the impact of full separation on calf behavior.