Background: Calorie restriction (CR) retards aging and increases longevity in many animal models. However, it is unclear whether CR can be implemented in humans without adverse effects on body composition. Objective: We evaluated the effect of a 2-y CR regimen on body composition including the influence of sex and body mass index (BMI; in kg/m(2)) among participants enrolled in CALERIE-2 (Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy), a multicenter, randomized controlled trial. Design: Participants were 218 nonobese (BMI: 21.9-28.0) adults aged 21-51 y who were randomly assigned to 25% CR (CR, n = 143) or ad libitum control (AL, n = 75) in a 2: 1 ratio. Measures at baseline and 12 and 24 mo included body weight, waist circumference, fat mass (FM), fat-free mass (FFM), and appendicular mass by dual-energy X-ray absorptiometry; activity-related energy expenditure (AREE) by doubly labeled water; and dietary protein intake by self-report. Values are expressed as means +/- SDs. Results: The CR group achieved 11.9% +/- 0.7% CR over 2-y and had significant decreases in weight (27.6 +/- 0.3 compared with 0.4 +/- 0.5 kg), waist circumference (-6.2 +/- 0.4 compared with 0.9 +/- 0.5 cm), FM (-5.4 +/- 0.3 compared with 0.5 +/- 0.4 kg), and FFM (-2.0 +/- 0.2 compared with -0.0 +/- 0.2 kg) at 24 mo relative to the AL group (all between-group P < 0.001). Moreover, FFM as a percentage of body weight at 24 mo was higher, and percentage of FM was lower in the CR group than in the AL. AREE, but not protein intake, predicted preservation of FFM during CR (P < 0.01). Men in the CR group lost significantly more trunk fat (P = 0.03) and FFM expressed as a percentage of weight loss (P < 0.001) than women in the CR group. Conclusions: Two years of CR had broadly favorable effects on both whole-body and regional adiposity that could facilitate health span in humans. The decrements in FFM were commensurate with the reduced body mass; although men in the CR group lost more FFM than the women did, the percentage of FFM in the men in the CR group was higher than at baseline.