Our objective was to study the effect of duration of milk accumulation on milk fat globules (MFG) secretion to better understand relationships between milk yield, milk fat, and MFG secretion. The modification of the milk accumulation duration in the udder is a tool to increase milk fat content. Four milking frequencies were studied on 6 dairy cows averaging 118 ± 22 d in milk: 2 milkings/d separated by 11- and 13-h or by 4- and 20-h intervals and 1 milking/d. The experimental trial was a double Latin square 3 × 3 with 2-wk periods. Postexperiment, a milking frequency of 36-h was repeated twice. Compared with 2 milkings with 11- and 13-h frequencies, 1 milking/d reduced milk and milk fat yields and increased fat content, without any effect on the size of MFG. Two milkings with 4- and 20-h intervals had no significant effect on milk fat yield and content but tended to increase the size of the MFG. Lipolysis, measured on morning milk, was weaker with 1 milking/d. When data were analyzed according to milk accumulation duration (4, 11, 13, 20, 24, and 36 h), the highest fat content and the largest diameters of MFG were obtained on milks from 4 and 36 h milkings (62.8 g/kg, 4.15 μm and 57.7 g/kg, 4.09 μm, respectively). Such observations could have 2 origins: the richness in residual milk of the 4-h milk and the coalescence of MFG related to the long milk accumulation duration in the 36-h milk. For each duration of milk accumulation, a relationship exists between MFG size and fat yield. The positive relation between MFG size and fat content was confirmed at each duration of milk accumulation. Rate of secretion of milk fat (milk accumulation of 4 h excluded) was also well correlated with MFG size. For the 36-h milk, this relationship was also observed but with a significantly different slope, assuming phenomena of MFG coalescence in response to the supposed increased intramammary pressure or to slower secretion rate and, hence, fusion events of microlipids droplets in the cytoplasm. Duration of milk accumulation joined with large increases in milk fat content induces changes in MFG size.