Six primiparous Holstein cows were assigned to a half udder design (n=6) 40 days prior to parturition. Beginning at parturition, one udder half was milked once daily (24hr interval) and the other four times daily (6hr interval). Udder halves were biopsied at days 15, 60, 120, and 230 of lactation for mammary tissue to perform mitochondrial staining and apoptosis studies. Increasing the milking frequency from 1x to 4x elevated the 4x udder half milk yield at early (d1-45) (P<0.0001), mid (d46-150) (P<0.0001) and late (d151-230) (P<0.0001) lactation. Milk protein percent (P= 0.013), lactose percent (P=0.004) and SNF percent (P=0.006), were elevated in milk from 4x udder halves over milk from 1x udder halves. We did not detect an effect of increased milking frequency on milk fat percent (P=0.25); however, yield of all components was increased. Increased milking frequency also increased mitochondrial numbers in mammary cells from 4x udder half (P=0.002) compared to 1x. We did not detect an effect of increased milking frequency on mammary apoptosis percentage. We also did not detect a difference in the abundance of gene transcripts for SOCS1, SOCS2, SOCS3 and CIS in milk; but could find an increase in alpha-lactalbumin (P=0.04) and beta-casein (P=0.001) 4x udder half gene transcripts.