To investigate whether short-term fasting affects serum testosterone (T) in normal subjects, 10 healthy men of normal weight were studied on two occasions: (1) after an overnight fast (8 h), and (2) after an additional 48 h of fasting. Blood glucose declined by 22 ± 3% between the tests (p < 0.001). Basal serum T fell from 8.7 ± 0.7 to 5.7 ± 0.8 µg/l (p < 0.01), and LH from 6.9 ± 0.8 to 5.0 ± 0.7 U/l (p < 0.01). Serum estradiol (E2) and FSH remained unaffected. To explore possible mechanisms behind the decreased basal release of T and LH, 9 small doses of glucose were given orally at regular intervals during a 56-hour fast to 9 additional normal men to maintain blood glucose levels. These men did not experience a fall in serum T or LH. Six additional normal men were given 50 µg GnRH intravenously after an overnight fast, and after a fasting period of 56 h. No acute increase in T was seen after the overnight fast, but after the 56-hour fast GnRH raised serum T by 55 ± 14% (p < 0.02). Moreover, fasting augmented the GnRH-induced LH response by 64 ± 15% (p < 0.02. These results imply that: (1) short-term fasting exerts inhibitory influence on Leydig cell function via a mechanism which might involve a reduced hypothalamic and/or pituitary stimulation; (2) this effect is blocked by oral glucose supplementation, and (3) the acute Leydig cell response to exogenous GnRH, which is seen after fasting, migh either reflect a changed release mechanism for stored LH, or up regulation of pituitary GnRH receptors during food deprivation.