Dairy cows usually store more milk in rear than in front quarters. At conventional machine milking this leads to an earlier cessation of milk flow in front than in rear quarters and hence unavoidable milking on empty front teats. We tested the hypothesis that an increased pulsation ratio in rear quarters reduces both milking on empty front teats and machine-on time. We have also tested if the different amplitude of cyclic vacuum fluctuations during milking with simultaneous vs. alternate pulsation within front and rear quarters, respectively, impacts milking characteristics. Ten Holstein dairy cows were milked twice daily at 14-h and 10-h milking intervals. Pulsation ratio in front quarters was 65:35, but was either set at 65:35, 70:30, 75:25 or 80:20 in rear quarters. Whole udder milk flow, claw vacuum, and mouthpiece chamber vacuum of all 4 quarters were recorded during milking. Teat tissue thickness of all 4 teats was measured with a cutimeter at 5 and 30 min after cluster detachment. Total milk yield did not differ among treatments, indicating complete udder emptying. The time of quarter milk flow (tQMF) of front quarters did not differ among treatments but decreased with increasing pulsation ratio in rear quarters. In addition, the faster milking of rear quarters at higher pulsation ratio caused higher peak and average milk flow rates, a shorter duration of milk flow decline, and a reduced machine-on time. The increased milk flow at a higher pulsation ratio did not cause a significant reduction of the claw vacuum at the used sample size. Teat tissue thickness did not significantly differ among treatments, likely because milking on empty teats did almost not occur in the rear quarters, and treatment settings did not differ in front quarters. Simultaneous pulsation caused higher amplitudes of cyclic vacuum fluctuation than alternate pulsation. However, milking characteristics did not differ between simultaneous and alternate pulsation. In conclusion, an increased pulsation ratio in rear quarters reduces machine-on time, increases milking performance, and reduces the time of milking on empty teats in front quarters, without an increased impact on teat tissue thickness.