Prestimulation administered to a cow before attachment of the milking unit has historically been performed manually. Development of innovative milking technology has allowed manual stimulation to be replaced by mechanical forms of stimulation. Holstein cows (n=30) were enrolled in a crossover design to determine the effect of manual stimulation (forestripping and drying) and high-vibration pulsation on oxytocin profiles, milk yield, milk flow rates, incidence of delayed milk ejection causing bimodal milk flow curves, and residual milk in Holstein cows milked 3 times daily (3×). All cows were subjected to all treatments. Cows received manual (forestripping and drying) or mechanical (high-vibration pulsation) stimulation along with lag times of 0, 30, or 90 s for 21 consecutive milkings. Forestripping involved the manual removal of 2 streams of milk from each teat and drying of the teats. High-vibration pulsation involved increasing the pulsation cycles from 60 to 300 pulses/min and reducing the vacuum in the pulsation chamber to 20 kPa. The 5 treatments were (1) immediate attachment of the milking machine under normal pulsation (T0); (2) dip plus forestrip and drying with 30-s lag time (FD30); (3) dip plus forestrip and drying with 90-s lag time (FD90); (4) high-vibration pulsation for 30 s (HV30); and (5) high-vibration pulsation for 90 s (HV90). Milk yield per milking averaged 14.0 kg and was significantly different among treatments; however, the maximum difference detected among treatments was 0.8 kg/milking. Milking unit on-time, which represents the time when the milking unit is under normal pulsation and harvesting milk (excluding the high-vibration pulsation time of 30 or 90 s), was shortest (245 s) for cows subjected to 90 s of high-vibration pulsation (HV90) and ranged from 256 to 261 s for all other treatments. Milk harvest may have begun during high-vibration pulsation; however, only 0.13 and 0.32 kg of milk was harvested during high-vibration pulsation for HV30 and HV90, respectively. The incidence of bimodal milk curves was lowest for FD90 (7%) and highest for T0 (21%). The somatic cell count was <72×10(3) cells/mL for all treatments. Residual milk obtained by giving 10 IU of oxytocin in the jugular vein followed by 2 min of milking unit attachment represented 12 to 14% of the total milk and did not differ among treatments. Endogenous oxytocin profiles peaked between 12.4 and 18.3 pg/mL for all treatments, and the peak occurred sooner in manually stimulated cows; however, we detected no difference in oxytocin concentration beyond 2 min after milking unit attachment. High-vibration pulsation elicited a similar oxytocin release when taking the start time of stimulation from manual stimulation or high vibration into consideration. Forestripping for visual observation of milk combined with the use of high-vibration stimulation may reduce variation in the milking routine. A predetermined lag time with minimal variation may be achieved via the time spent in high-vibration stimulation instead of a lag period...