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The role of nutrition in modulating postexercise overnight recovery remains to be elucidated. We assessed the effect of protein ingestion immediately before sleep on digestion and absorption kinetics and protein metabolism during overnight recovery from a single bout of resistance-type exercise.Sixteen healthy young males performed a single bout of resistance-type exercise in the evening (2000 h) after a full day of dietary standardization. All subjects were provided with appropriate recovery nutrition (20 g of protein, 60 g of CHO) immediately after exercise (2100 h). Thereafter, 30 min before sleep (2330 h), subjects ingested a beverage with (PRO) or without (PLA) 40 g of specifically produced intrinsically [1-13C]phenylalanine-labeled casein protein. Continuous intravenous infusions with [ring-2H5]phenylalanine and [ring-2H2]tyrosine were applied with blood and muscle samples collected to assess protein digestion and absorption kinetics, whole-body protein balance and mixed muscle protein synthesis rates throughout the night (7.5 h).During sleep, casein protein was effectively digested and absorbed resulting in a rapid rise in circulating amino acid levels, which were sustained throughout the remainder of the night. Protein ingestion before sleep increased whole-body protein synthesis rates (311 ± 8 vs 246 ± 9 μmol·kg−1 per 7.5 h) and improved net protein balance (61 ± 5 vs −11 ± 6 μmol·kg−1 per 7.5 h) in the PRO vs the PLA experiment (P < 0.01). Mixed muscle protein synthesis rates were ∼22% higher in the PRO vs the PLA experiment, which reached borderline significance (0.059%·h−1 ± 0.005%·h−1 vs 0.048%·h−1 ± 0.004%·h−1, P = 0.05).This is the first study to show that protein ingested immediately before sleep is effectively digested and absorbed, thereby stimulating muscle protein synthesis and improving whole-body protein balance during postexercise overnight recovery.