Fat mobilization in adipose tissue (AT) has a specific timing. However, circadian rhythms in the activity of the major enzyme responsible for fat mobilization, Hormone-Sensitive Lipase (HSL) have not been demonstrated in humans.To analyze in a cross-sectional study, whether there is an endogenous circadian rhythm in HSL activity in human AT ex vivo and whether rhythm characteristics are related to food-timing or fasting-duration.Abdominal AT biopsies were obtained from eighteen severely-obese participants (age:46±11y; BMI 42±6 kg/m2) who underwent laparoscopic-gastric-bypass. 24-h rhythms of HSL activity and LIPE (HSL transcript in humans) expression in subcutaneous AT were analyzed together with habitual food-timing and night-fasting-duration.HSL activity exhibited a circadian rhythm (P=0.023) and reached the maximum value at Circadian Time 16 (CT) that corresponded to around midnight (relative local clock time. Similarly, LIPE displayed a circadian rhythm with acrophase also at night (P=0.0002). Participants with longer night-fasting-duration > 11.20 h displayed almost double the amplitude (1.91 times) in HSL activity rhythm than those with short duration (P=0.013); while habitual early diners (before 21:52 h) had 1.60 times higher amplitude than late diners (P=0.035).Our results demonstrate circadian rhythms in HSL activity and may lead to a better understanding of the intricate relationships between food-timing, fasting-duration, and body fat regulation.