Normal -cells adjust their function to compensate for any decrease in insulin sensitivity. Our aim was to explore whether a prolonged fast would allow a study the effects of changes in circulating free fatty acid (FFA) levels on insulin secretion and insulin sensitivity, and whether any potential effects could be reversed by the anti-lipolytic agent Acipimox. 14 (8 female, 6 male) healthy young adults (aged 22.8-26.9 years) without a family history of diabetes and a BMI of 22.6±3.2 kg/m² were studied on three occasions in random order. Growth hormone and FFA levels were regularly measured overnight (22:00-07:59), and subjects underwent an intravenous glucose tolerance test (IVGTT) in the morning (08:00-11:00) on each visit. Treatment A was an overnight fast, treatment B was a 24-hour fast with regular administrations of a placebo and treatment C was a 24-hour fast with regular ingestions of 250 mg of Acipimox. The 24-hour fast increased overnight FFA levels (as measured by the AUC) 2.8-fold (51.3 [45.6-56.9] vs. 18.4 [14.4-22.5] *10⁴ µmol/l*min, p<0.0001), and led to decreases in insulin sensitivity (5.7 [3.6-8.9] vs. 2.6 [1.3-4.7] *10^-4 min^-1 per mU/l, p<0.0001) and the acute insulin response (16.3 [10.9-21.6] vs. 12.7 [8.7-16.6] *10² pmol/l*min, p=0.02), and therefore a reduction in the disposition index (93.1 [64.8-121.4] vs. 35.5 [21.6-49.4] *10² pmol/mU, p<0.0001). Administration of Acipimox during the 24-hour fast lowered FFA levels by an average of 20% (range: -62% to +49%, p=0.03), resulting in a mean increase in the disposition index of 31% (p=0.03). The 24-hour fast was accompanied by substantial increases in fasting NEFA levels, and induced reductions in the acute glucose-simulated insulin response and insulin sensitivity. The use of Acipimox during the prolonged fast increased the disposition index, suggesting a partial reversal of the effects of fasting on the acute insulin response and insulin sensitivity.