Increased milking frequency (IMF) in early lactation is a time and cost-effective farm management practice to enhance profit in the dairy industry. The process involves milking cows more often in early lactation alone. On farms that milk twice daily (2X), early lactation cows are milked four times daily (4X) for 21 d postpartum. Cows produce significantly more milk during this timeframe and continue to have increased milk yield (MY) when returned to 2X milking for the remainder of lactation. The objective of this dissertation was to discover the physiological processes of early lactation IMF that cause increased MY throughout lactation. All studies involved unilateral frequent milking (UFM) with 2X and 4X udder halves for 21 d in early lactation. The first study manipulated milking interval (MI), or the time between milkings, during early lactation IMF. Cows were either milked on an even MI every 6 h or unevenly on a 9:3:9:3 h MI. Unevenly milked cows produced more milk on the final day of 4X treatment, but there was no significant difference in the increased MY carry-over effect between MI groups. Therefore, farmers can utilize any MI that fits their schedule and still achieve significantly enhanced profits. The second study aimed to infer the metabolic mechanisms of early lactation IMF that increase MY by comparing it to bovine somatotropin (bST). Cows that underwent early lactation IMF received bST at 80 DIM. Both IMF and bST treatments significantly enhanced MY, but there was no interaction or synergistic effect between treatments. We surmised that IMF and bST cause increased MY through different metabolic mechanisms since IMF functions locally and bST operates systemically. The final study analyzed mammary tissue from 2X and 4X udder halves on the final day of UFM treatment. The mechanism in which IMF enhanced MY involved increased protein levels of signal transducer and activator of transcription (STAT5), activated and total protein kinase B (Akt), and total extracellular signal-regulated kinase 1 and 2 (ERK1/2) and reduced protein levels of total mammalian target of rapamycin (mTOR) and total mitogen activated protein kinase (MAPK) in 4X udder halves compared to 2X. / Doctor of Philosophy / Increased milking frequency in early lactation is the process of milking cows more often for the first 3 weeks after calving. Cows not only produce more milk during this timeframe but continue to have elevated milk yield throughout lactation. This phenomenon is called the increased milk yield carry-over effect. This dissertation aimed to further enhance the increased milk yield carry-over effect of early lactation increased milking frequency. All studies utilized unilateral frequent milking with left udder halves milked twice daily and right udder halves milked four-times daily. The first study compared even and uneven milking intervals alongside early lactation increased milking frequency. The right udder halves of the even groups were milked every 6 hours. Cows in the uneven milking interval group were milked on a 9:3:9:3 hour interval. The uneven milking interval group produced more milk than even milking interval group on the final day of milking frequency treatment. However, there was no difference in milk yield between groups at any other time point. All cows had an increased milk yield carry-over effect throughout 300 days of lactation. In the second study, cows received bovine somatotropin in mid-lactation to observe possible synergistic effects in enhancing milk yield after early lactation increased milking frequency. Both the bovine somatotropin and increased milking frequency increased milk yield, but there was no synergistic effect when the two treatments were combined. The final study analyzed proteins within mammary tissue after 3 weeks of unilateral frequent milking. Udder halves milked four-times daily had significantly elevated activated and total STAT5, activated and total Akt, and total ERK1/2. Udder halves milked twice daily had elevated total MAPK and total mTOR. These findings helped to understand the metabolic functioning of increased milking frequency in early lactation that causes a persistent increase in milk yield throughout lactation.