Increasing the milking interval decreases milk yield and modifies milk composition. To gain a clearer understanding of the regulation of milk yield and composition, a study was conducted to establish the response curves of nutrient extraction by the mammary gland and mammary epithelial permeability in response to increasing milking intervals. Four multiparous lactating dairy cows were milked at 8-, 12-, 16-, or 24-h intervals over a period of 7 d using a Latin square design. Between the 8- and 24-h milking intervals, milk yield and milk protein levels fell curvilinearly from 38.2 to 29.2 kg/d and from 1,086 to 827 g/d, respectively. Milk fat yield decreased linearly from 1,475 to 1,235 g/d. Indicators of the opening of tight junctions increased linearly with increasing milking intervals: milk BSA increased from 148 to 207 mg/L and plasma lactose increased from 22.9 to 32.0 mg/L. The mammary gland's ability to extract nutrients decreased with increasing milking intervals. Extraction rates of glucose, beta-hydroxybutyrate, and total glycerol decreased significantly (from 27.2 to 23.3%, from 42.3 to 34.4%, from 36.6 to 30.8% between 8- and 24-h milking intervals, respectively), and not significantly for alpha-amino nitrogen (from 23.2 to 20.0%). The extraction rate of acetate remained constant. Moreover, the extraction of milk fat precursors appeared to be less regulated than those of the precursors of milk protein and lactose, which could partly explain why milk yield and milk protein yield decreased more than milk fat yield. The arteriovenous differences of beta-hydroxybutyrate and total glycerol remained constant, whereas those of glucose decreased significantly from 0.98 to 0.87 +/- 0.05 mmol/L and not significantly from 0.74 to 0.64 +/- 0.12 mmol/L for alpha-amino nitrogen. As a result, the mammary gland's ability to extract nutrients appears to be downregulated explaining partly the decrease in daily milk yield observed in response to increased milking intervals.