We compared within and between breastfeed changes in milk fat to short-term rates of milk synthesis and degree of breast emptying (measured using the Computerized Breast Measurement system) over two 24 h periods for five lactating mothers. The fat content (f) of fore and hind milk samples increased more steeply as the breast was progressively emptied by the infant (degree of emptying, d, range 0-1; f = 21.59 + 9.38d + 70.99d2; P < 0.0001; r2 = 0.68; n = 154). For the nine individual breasts, between 41-95% of the variance of the fat content of milk was explained by degree of breast emptying. We argue that this relationship explains differences in the circadian rhythm of the fat content of milk and allows the accurate calculation of the average fat content of milk consumed by infants (37-66 g/l for the nine individual breasts). The fatty acid composition of the fore and hind milk samples was determined for four of the mothers. We observed within and between breastfeed variability in the relative proportions of the seven major fatty acids of milk fat and these changes are discussed with reference to the control of fat synthesis in the human mammary gland.