Normal β-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 of 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 antilipolytic agent acipimox. Fourteen (8 female, 6 male) healthy young adults (aged 22.8–26.9 yr) without a family history of diabetes and a body mass index of 22.6 � 3.2 kg/m² were studied on three occasions in random order. Growth hormone and FFA levels were regularly measured overnight (2200-0759), and subjects underwent an intravenous glucose tolerance test in the morning (0800-1100) on each visit. Treatment A was an overnight fast, treatment B was a 24-h fast with regular administrations of a placebo, and treatment C was a 24-h fast with regular ingestions of 250 mg of acipimox. The 24-h fast increased overnight FFA levels (as measured by the area under the curve) 2.8-fold [51.3 (45.6–56.9) vs. 18.4 (14.4–22.5) *10⁴ μmol/l*min, P < 0.0001], and it 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-h 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). In conclusion, the 24-h fast was accompanied by substantial increases in fasting FFA 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.