The purpose of this study was to determine the impact of ingesting 30 g casein protein with and without 2 g free leucine before sleep on myofibrillar protein synthesis rates during postexercise overnight recovery. Thirty-six healthy young men performed a single bout of resistance-type exercise in the evening (1945) after a full day of dietary standardization. Thirty minutes before sleep (2330), subjects ingested 30 g intrinsically l-[1-¹³C]phenylalanine-labeled protein with (PRO+leu, n = 12) or without (PRO, n = 12) 2 g free leucine, or a noncaloric placebo (PLA, n = 12). Continuous intravenous l-[ ring-²H₅]phenylalanine, l-[1-¹³C]leucine, and l-[ ring-²H₂]tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole body protein net balance, myofibrillar protein synthesis rates, and overnight incorporation of dietary protein-derived amino acids into myofibrillar protein. Protein ingestion before sleep improved overnight whole body protein net balance ( P < 0.001). Myofibrillar protein synthesis rates did not differ significantly between treatments as assessed by l-[ ring-²H₅]phenylalanine (0.057 ± 0.002, 0.055 ± 0.002, and 0.055 ± 0.004%/h for PLA, PRO, and PRO+leu, respectively; means ± SE; P = 0.850) or l-[1-¹³C]leucine (0.080 ± 0.004, 0.073 ± 0.004, and 0.083 ± 0.006%/h, respectively; P = 0.328). Myofibrillar l-[1-¹³C]phenylalanine enrichments increased following protein ingestion but did not differ between the PRO and PRO+leu treatments. In conclusion, protein ingestion before sleep improves whole body protein net balance and provides amino acids that are incorporated into myofibrillar protein during sleep. However, the ingestion of 30 g casein protein with or without additional free leucine before sleep does not increase muscle protein synthesis rates during postexercise overnight recovery.