Background: Leptin is involved in the regulation of eating behavior. Its serum levels are determined by fat mass but a diurnal rhythm is also described. It is not clear whether leptin levels are also controlled in vivo by hormonal stimuli, like insulin or cortisol. Methods and Results: This possible temporal relation was investigated by serial measurements during 24 h (group A) and 46 h (group B) in 15 healthy volunteers and another 10 subjects (group C) while fasting for 72 h. Maximal leptin levels were observed at 4:00 a.m. and 4:00 p.m. in subjects on a normal diet. During 24 h starvation (group B), there was a 40% decrease of mean leptin concentration when compared to baseline values. In group C, the leptin concentration under starvation dropped to 25% of basal levels after 72 h. Pooled data from group A and the nonfasting data from group B showed an insulin increase preceding leptin increase by 6 h (r = 0.405, p < 0.0001), while cortisol decreased 4 h (r = 0.361, p < 0.001) after leptin decrease. Conclusion: Starvation results in a fall of circulating leptin, ending leptin rhythmicity. Food intake is causally involved in the fluctuation of leptin levels in serum. Presumably this effect is mediated by insulin, while cortisol does not seem to affect leptin release directly in vivo.