The purpose of this study was to determine the impact of ingesting 30 g casein protein with and without 2 g free leucine prior to sleep on myofibrillar protein synthesis rates during post-exercise overnight recovery. 36 healthy young males performed a single bout of resistance-type exercise in the evening (19:45 h) after a full day of dietary standardization. Thirty min prior to sleep (23:30 h), 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 prior to 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^-1 for PLA, PRO, and PRO+leu, respectively; P=0.850) or L-[1-¹³C]-leucine (0.080±0.004, 0.073±0.004, and 0.083±0.006 %•h-1, 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 prior to 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 prior to sleep does not increase muscle protein synthesis rates during post-exercise overnight recovery.