The present study tested the hypothesis that supplemental dietary fatty acids (FA) affect the energy corrected milk yield in proportion to the milk production level of dairy cows, and increase both long chain FA proportion of milk FA and milk fat globule diameter. Sixteen Danish Holstein cows were divided into four 4x4 Latin squares with two squares of medium yielding cows (32.2 kg energy corrected milk (ECM)/d; 158 days in milk (DIM)) and two squares of high yielding cows (40.0 kg ECM/d; 74 DIM). Experimental length was 12 weeks, with three weeks for each of the four periods. The four treatments were no supplementation (17 g FA/kg dry matter (DM)) and three diets with supplemented FA (29, 40, and 52 g total FA/kg DM, respectively) obtained by substituting barley with Palm Fatty Acid Distillate (PFAD) fat. Diets were offered as total mixed rations with 63% grass/clover silage (DM basis). Dry matter intake decreased with increasing FA supplementation, but net energy intake was not affected. The general linear responses to 10 g/kg DM increase in FA level were 1.1 kg ECM (P<0.0001), 0.061 kg milk fat (P<0.0001), 0.012 kg milk protein (P=0.09) and 0.052 kg lactose (P=0.0002) per day, and linear responses in milk composition were 0.39 g fat (P=0.07), -0.71 g protein (P<0.0001) and 0.05 g lactose (P=0.3) per kg milk, and 0.092 microm (P<0.0001) in milk fat average globule diameter. Fatty acid supplementation decreased short- and medium-chain FA and C16:0 and increased C18:1 proportions of total FA in milk. Supplemental dietary FA increased ECM yield but not in proportion to production level as anticipated, and increased average FA chain length and milk fat globule diameter.