The aim of the present study was to study the effect of milking cows 4 times daily on free fatty acids (FFA) in the milk compared with milking twice daily. An experiment was performed during 2 wk in which half udders in 11 cows were milked 2 or 4 times daily. Milk yield was measured, and milk was analyzed for fat content, FFA, fatty acid composition, fat globule size, and activity of gamma-glutamyl transpeptidase. Concentration of FFA was greater (1.49 mEq/100 g of fat) in milk from half udders milked 4 times daily than in milk from the half udders milked twice daily (1.14 mEq/100 g of fat). Further, it was noted that milk from the half udder milked 4 times daily contained milk fat globules with larger average diameters. Increased milking frequency increased milk yield by 9% compared with the udder half milked twice daily, but fat content and fat yield were not affected. The results are of importance for further understanding the mechanisms behind the increased content of FFA that is frequently observed in automatic milking systems.