Milk lipolysis is defined as the hydrolysis of triglycerides, which are the main component of milk fat. Short-chain fatty acids (FAs) released in milk are responsible for rancid flavour. In addition, the presence of partial glycerides impairs the functional properties of milk, such as foaming and creaming abilities. Milk lipolysis, a key criterion used to assess milk quality, depends on animal parameters and breeding factors. Low-energy diets are associated with higher levels of spontaneous lipolysis, particularly in late lactation. In this study, dairy cows were fed a restricted diet (i.e. 65% of their ad libitum DM intake (DMI)) to induce spontaneous lipolysis in milk and to study milk composition associated with lipolysis. Two groups of 22 cows each received a control diet (100% of ad libitum DMI) or the restricted diet according to a 2 x 2 crossover design. The restricted diet was fed for five days. As expected, feed restriction increased milk spontaneous lipolysis which was associated with an increase in lipoprotein lipase activity. At the same time, milk yield and protein content decreased and no effect was observed on milk fat content. The increase in spontaneous lipolysis was associated with an increase in milk fat globules diameter, without influencing casein micelles diameter. Feed restriction altered the parameters of dairy cow metabolism, with increases in plasma non-esterified FAs, triglycerides and urea, indicating body fat mobilisation and protein catabolism associated with feed restriction. Feed restriction also altered hormonal parameters, with decreases in plasma insulin, insulin-like growth factor 1 and prolactin. As expected, lipolysis was higher in evening milk and was associated with a larger diameter of milk fat globules. This zootechnical approach will be completed with proteomic, lipidomic and transcriptomic studies of milk and/or mammary gland of animals selected for their extreme lipolysis.