It has been well established that milk yield is affected both by milking frequency and due to the removal of residual milk, but the influence of a combination of these factors is unclear. In this study, four mid-lactation cows were used in a 4 × 4 Latin square design to test the hypothesis that the effects of more frequent milking and residual milk removal on milk yield and composition are additive and alter milk fatty acid composition. Treatments comprised two or four times daily milking in combination with (or without) residual milk removal over a 96 h interval preceded by a 2 d pretreatment period and followed by a 8 d washout in each 14 d experimental period. Milk was sampled at each milking for the analysis of gross composition and SCC. Samples of available and residual milk collected on the last milking during each treatment period were collected and submitted for fatty acid composition analysis. Increases in milking frequency and residual milk removal alone or in combination had no effect on milk yield or on the secretion of lactose and protein in milk. However, residual milk removal during more frequent milking increased milk fat yield. Milking treatments had no major influence on the fatty acid composition of available milk, but resulted in rather small changes in the relative abundance of specific fatty acids, with no evidence that the additive effects of treatments were due to higher utilisation of preformed fatty acids relative to fatty acid synthesis de novo. For all treatments, fat composition of available and residual milk was rather similar indicating a highly uniform fatty acid composition of milk fat within the mammary gland.