Increasing the milking frequency of early lactation dairy cows increases their milk yield, both during increased milking and after the cows are returned to a normal twice-daily milking schedule. When milked four times a day (4X) for only the first twenty-one days of lactation, the right udder half produces 3 kg/d more milk than the left half, which is milked only two times a day (2X) over the course of lactation (Hale et al., 2003). Alterations to this increased milking frequency (IMF) approach have been investigated in order to maximize production of the animals and determine the most efficient practice for producers. The aim of this study was to determine the appropriate duration of early lactation IMF treatment by increasing milking frequency of early lactation cows for various lengths of time, and subsequently increasing the use of this management practice on Virginia dairy farms. The right udder half of twenty-three primiparous and multiparous Holstein cows were milked 4X for 10, 20, or 40 days at the beginning of lactation, and the left udder half 2X for the entire lactation. Udder-half milk yields were measured at various time points throughout lactation and used to calculate the difference between right (4X) and left (2X) udder halves. Overall, treatment did not have a significant effect on milk yield difference throughout the entire lactation; the udder half differences for each group were -0.45 kg, 1.92 kg and 4.62 kg for the 10 d, 20 d and 40 d treatments (P > 0.05). In addition to the IMF portion of the experiment, two different methodologies were used to investigate the possible mechanism of local regulation of milk yield in response to IMF treatment. Milk fatty acid analysis was performed on milk samples obtained from the above experiment. Three different groups of fatty acids were analyzed to detect potential changes in the right udder half (4X) when compared to the left (2X); the three groups were denovo, C16, and preformed fatty acids. There was no significant effect of treatment on fatty acid composition of right and left udder halves for any of the three groups (P > 0.05). The second methodology used to explore a possible mechanism behind increased milk yield following IMF treatment was immunohistochemistry of mammary gland tissue samples obtained after IMF treatment in a previous experiment. The key target investigated was a component of the Hippo signaling pathway, Yes-associated protein (YAP). Intensity of YAP staining in the cytoplasmic area of mammary epithelial cells (MEC) and number of YAP-positive stained nuclei located in the MEC were quantified for each of the images obtained. There was no effect of treatment or day on intensity of staining (P > 0.05) with no difference in the intensity of staining between 4X and 2X samples or d 21 and d 60 samples. However, the interaction for treatment x day tended to be significant (P < 0.06), with the d 60 samples tending to have higher intensity of staining than d 21 samples. For YAP-positive nuclei, there was a significant effect of day (P < 0.05), with d 60 samples having significantly more YAP positive nuclei. There was not a significant effect of treatment or treatment x day interaction (P > 0.05) with 2X and 4X samples having the same number of YAP positive nuclei. Results from the first experiment reveal that 40 d of IMF during early lactation is sufficient to produce an increase in milk and component yields throughout lactation. This practice could be implemented on Virginia dairy farms as a way to increase efficiency and milk yield per cow. Results from the second half of this research indicates that further research is needed to investigate the fatty acid content of milk from cows subject to IMF treatment during early lactation. In addition, YAP potentially plays a role in the changes occurring in the mammary gland, with increased intensity of YAP staining and increased number of YAP positive nuclei observed at 60 DIM. Understanding of this protein and its involvement in the mammary gland could lead to identifying a mechanism for which this increase in milk yield and components following IMF is occurring. Further research needs to be done to provide results supporting the current experiment.