Milk lipolysis is defined as the hydrolysis of triglycerides, the major component of milk fat, resulting in the release of short-chain fatty acids (FA) responsible for rancid flavor and partial glycerides that impair functional properties such as foaming and creaming abilities. Milk lipolysis is a complex phenomenon that depends on both animal parameters and breeding factors. Milk spontaneous lipolysis is known to be higher in milk from evening milkings than from morning milkings. This may be related to the longer length of overnight milking intervals or to the nycthemeral cycle. In this experiment, our objective was thus to study the impact of both milking intervals and time of day on milk spontaneous lipolysis in twice-daily-milking systems with one of 3 milking intervals: Short Day - Long Night (SD-LN, 6.30 a.m. and 4.30 p.m.,); Long Day - Short Night (LD-SN, 6:30 a.m. and 8:30 p.m.,); and Balanced Day and Night (BDN, 6:30 a.m. and 6:30 p.m.,). To achieve this goal, 21 multiparous dairy cows in mid-lactation were used in a 3 * 3 Latin square design over 3 periods. The experiment lasted 5 weeks, corresponding to 3 experimental periods of 6 d alternating with 8 d of milking with conventional hours (morning-evening gap of 10 h). We confirmed that milk spontaneous lipolysis was influenced by milking interval, but not the milking time. Indeed, we observed more lipolysis in SD-LN evening milk (+0.20 mEq/100 g fat) and LD-SN morning milk (+0.22 mEq/100 g fat), both of which corresponded to a 10 h interval between successive milkings. High lipolysis milk came from cows that produced less milk with a higher milk fat content. No significant difference between milkings was observed for BDN. Milk protein, total P and citrate contents increased according to the duration of mammary gland storage of milk (from 10 to 14 h). There was no effect of milking intervals on milk fat globule diameter. The milk Na+/K+ ratio, indicating an opening of tight junctions in the mammary gland, increased only in evening milkings with BDN and LD-SN. In conclusion, we found that the effect of milking intervals on lipolysis is stronger than that of the nycthemeral cycle.