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BACKGROUND: Muscle disuse leads to muscle atrophy that has been attributed to declines in basal and postprandial muscle protein synthesis (MPS) rates. Leucine regulates MPS and may attenuate disuse-induced declines in MPS rates and muscle mass. OBJECTIVES: The purpose of this study was to evaluate the capacity of leucine supplementation to attenuate disuse-induced declines in MPS rates and muscle mass in young and older adults. METHODS: In a randomized, double-blind, parallel-group design, 24 young (23 ± 4 y) and 24 older (69 ± 4 y) recreationally active adults (equal sex distribution) underwent 3 d of unilateral knee immobilization (leg casting) and received a leucine [group of adult study participants who supplemented with 5 g of leucine 3 × daily with each main meal during 3 d of unilateral knee immobilization by means of a full leg cast (LEU)] or energy-matched carbohydrate [group of adult study participants who supplemented with 5 g of carbohydrate 3 × daily with each main meal during 3 d of unilateral knee immobilization by means of a full leg cast (PLA)] supplement. Preimmobilization and postimmobilization, quadriceps muscle cross-sectional area (CSA) was assessed in the immobilized (IM) and nonimmobilized (NO-IM) leg by computed tomography. MPS rates were assessed in both legs during immobilization via HO coupled with saliva, blood, and muscle biopsy sampling. RESULTS: In young and older adults, MPS rates were ∼15% and ∼23% lower in the IM compared with NO-IM leg (1.28 ± 0.29 compared with 1.50 ± 0.26 and 1.10 ± 0.16 compared with 1.46 ± 0.28%/d, respectively; leg: both P < 0.001), with no differences between LEU compared with PLA treatments (treatment: P = 0.932 and P = 0.742, respectively). CSA decreased by ∼1.2% and ∼1.1% in the IM leg in young and older adults (from 7162 ± 1148 to 7076 ± 1129 mm and from 5813 ± 1092 to 5750 ± 1096 mm, respectively; leg × time interaction: both P < 0.001), with no differences between LEU compared with PLA (treatment: P = 0.374 and P = 0.998). IM leg MPS rates were lower in older compared with young adults [difference: -0.18 (95% confidence interval: -0.31, -0.04) %/d; P = 0.013]. No differences were observed in the absolute (mm) or relative (%) decline in CSA between young and older adults (both P > 0.05). CONCLUSIONS: Leucine supplementation does not attenuate the decline in daily MPS rates or muscle mass during short-term limb immobilization in young or older adults. Clinical Trial Register No. (Netherlands Trial Register): NL-OMON45771.