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Intermittent fasting may impart metabolic benefits independent of energy balance by initiating fasting-mediated mechanisms. This randomized controlled trial examined 24-hour fasting with 150% energy intake on alternate days for 3 weeks in lean, healthy individuals (0:150; n = 12). Control groups involved a matched degree of energy restriction applied continuously without fasting (75% energy intake daily; 75:75; n = 12) or a matched pattern of fasting without net energy restriction (200% energy intake on alternate days; 0:200; n = 12). Primary outcomes were body composition, components of energy balance, and postprandial metabolism. Daily energy restriction (75:75) reduced body mass (-1.91 ± 0.99 kilograms) almost entirely due to fat loss (-1.75 ± 0.79 kilograms). Restricting energy intake via fasting (0:150) also decreased body mass (-1.60 ± 1.06 kilograms; P = 0.46 versus 75:75) but with attenuated reductions in body fat (-0.74 ± 1.32 kilograms; P = 0.01 versus 75:75), whereas fasting without energy restriction (0:200) did not significantly reduce either body mass (-0.52 ± 1.09 kilograms; P ≤ 0.04 versus 75:75 and 0:150) or fat mass (-0.12 ± 0.68 kilograms; P ≤ 0.05 versus 75:75 and 0:150). Postprandial indices of cardiometabolic health and gut hormones, along with the expression of key genes in subcutaneous adipose tissue, were not statistically different between groups ( P > 0.05). Alternate-day fasting less effectively reduces body fat mass than a matched degree of daily energy restriction and without evidence of fasting-specific effects on metabolic regulation or cardiovascular health., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)