Ultimately milk yield is a function of the number of functional secretory cells in the mammary tissue and the metabolic activity of these cells. Both the number of cells and the cell activity are not static, but change during the course of lactation. The rate at which both cell number and activity change may be influenced by farm management practices, such as feeding, photoperiod, hormonal treatment (e.g. BST), and milking frequency or interval. By applying such practices, or a combination there of, at any time during the lactation the farmer has tools to alter the shape of the lactation curve. For instance, these practices could be applied following peak lactation to increase milk yield and perhaps slow down the rate of post-peak decline in milk yield, i.e. increase persistency, but can also be used to accelerate the post-peak decline to promote involution of the gland near the time of drying-off. The present review will focus on only one of these tools, i.e. milking frequency. Compared to a standard twice-daily milking regime, milking three times a day or more often (robotic milking) will increase milk yield by approximately 15%, whereas going from twice to once-daily can decrease milk output anywhere between 10 to 50%. Although more frequent milking is practised more often than once daily, the once-daily milked gland provides an excellent model to study functional changes related to milking interval. The effects of increasing and decreasing milking frequency or milking interval on mammary function will be reviewed at the whole gland as well as at the cellular level, with the emphasis being on functional changes in the once-daily milked gland with regard to processes such as changing mammary cell number and activity, feedback-inhibition, mammary tight junction leakiness, apoptosis and cisternal-alveolar milk storage.