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Protein ingestion and exercise stimulate myofibrillar protein synthesis rates. When combined, exercise further increases the postprandial rise in myofibrillar protein synthesis rates. It remains unclear whether protein ingestion with or without exercise also stimulates muscle connective tissue protein synthesis rates. The authors assessed the impact of presleep protein ingestion on overnight muscle connective tissue protein synthesis rates at rest and during recovery from resistance-type exercise in older men. Thirty-six healthy, older men were randomly assigned to ingest 40 g intrinsically L-[1-C-13]-phenylalanine and L-[1-C-13]-leucinelabeled casein protein (PRO, n = 12) or a nonprotein placebo (PLA, n = 12) before going to sleep. A third group performed a single bout of resistance-type exercise in the evening before ingesting 40 g intrinsically-labeled casein protein prior to sleep (EX+PRO, n = 12). Continuous intravenous infusions of L-[ring-H-2(5)]-phenylalanine and L-[1-C-13]-leucine were applied with blood and muscle tissue samples collected throughout overnight sleep. Presleep protein ingestion did not increase muscle connective tissue protein synthesis rates (0.049 +/- 0.013 vs. 0.060 +/- 0.024%/hr in PLA and PRO, respectively; p = .73). Exercise plus protein ingestion resulted in greater overnight muscle connective tissue protein synthesis rates (0.095 +/- 0.022%/hr) when compared with PLA and PRO (p < .01). Exercise increased the incorporation of dietary protein-derived amino acids into muscle connective tissue protein (0.036 +/- 0.013 vs. 0.054 +/- 0.009 mole percent excess in PRO vs. EX+PRO, respectively; p < .01). In conclusion, resistance-type exercise plus presleep protein ingestion increases overnight muscle connective tissue protein synthesis rates in older men. Exercise enhances the utilization of dietary protein-derived amino acids as precursors for de novo muscle connective tissue protein synthesis during overnight sleep.