The aim of this study was to investigate the effects of milking frequency reduction and dietary lipid supplementation on intake, BW, and milk yield and composition in high yielding dairy ewes. Ten lactating Assaf ewes were allocated into 2 experimental groups (n=5). Ewes were fed alfalfa hay ad libitum and 34 g·kg(-1) of BW of a concentrate feed with either 0 (Control group) or 43 g of sunflower oil·kg(-1) of DM (SO group). The experiment lasted 63 d and consisted of 3 periods. During Period 1 (from d 1 to 21), ewes were milked twice a day. During Period 2 (from d 22 to 49), ewes were unilaterally milked, so that each gland of each ewe was milked either once or twice daily. During Period 3 (from d 50 to the end of the experiment), both udder halves were again milked twice daily. Intake, BW, and milk composition were controlled weekly and milk production from each half udder was recorded twice a week. Total DM intake, BW, and milk yield in Period 1 were not significantly (P>0.10) affected by dietary treatments. Milk yield tended to be increased in the ewes fed the SO diet in periods 2 (P=0.093) and 3 (P=0.067). Oil supplementation (SO diet) significantly (P<0.05) decreased milk protein and total solids concentrations in the 3 experimental periods and fat content in Period 3, and tended (P=0.077) to decline fat content in Period 2. Lactose content and somatic cell count (SCC) were unaffected (P>0.10) by dietary lipid supplementation in any of the experimental periods. There were no significant (P>0.10) differences between half udders in milk yield and composition in Period 1, and in SCC in any of the experimental periods. Fat and total solids contents were unaffected (P>0.10) by reducing milking frequency. Nevertheless, milk protein content was increased (P<0.001) when glands were milked only once daily whereas milk yield and lactose content were decreased (P=0.001). The interaction between gland and diet was significant for lactose in Period 2, suggesting a greater effect of milking frequency reduction on tight junctions in Control ewes. In fact, the ratio between glands for milk yield was significantly (P<0.05) greater in SO (0.82) than in Control (0.72) ewes. In Period 3, this ratio increased but it was still lower in Control ewes (0.92 vs. 0.78, P<0.05). Thus, milking frequency reduction and SO supplementation seem to have counteracting effects on milk production and composition. Our results suggest that SO-supplemented ewes have a better capacity of adaptation to changes in milking frequency, probably due to processes induced in the mammary gland.