This research was carried out to quantify the effects of a range of variables on milk fat globule (MFG) size for a herd of Holstein-Friesian cows managed through an automatic milking system with year-round calving. We hypothesised that the overall variation in average MFG size observed between individual animals of the same herd cannot sufficiently be explained by the magnitude of the effects of variables that could be manipulated on-farm. Hence, we aimed to conduct an extensive analysis of possible determinants of MFG size, including physiological characteristics (parity, days in milk, days pregnant, weight, age, rumination minutes, somatic cell count) and milk production traits (number of milkings, milk yield, fat yield, protein and fat content, fat-protein ratio) on the individual animal level; and environmental conditions (diet, weather, season) for the whole herd. Our results show that when analysed in isolation, many of the studied variables have a detectable effect on MFG size. However, analysis of their additive effects identified days in milk, parity and milk yield as the most important variables. In accordance with our hypothesis, the estimated effects of these variables, calculated using a multiple variable linear mixed model, do not sufficiently explain the overall variation between cows, ranging from 2.70 to 5.69 µm in average MFG size. We further show that environmental variables, such as sampling day (across seasons) or the proportion of pasture and silage in the diet, have limited effects on MFG size and that physiological differences outweigh the effects of milk production traits and environmental conditions. This presents further evidence that the selection of individual animals is more important than the adjustment of on-farm variables to control MFG size.