The effect of a 4-wk aerobic exercise training program (30-45 min, 3-5 d/wk, >or=65% maximal heart rate) on mixed skeletal muscle protein fractional synthetic rate (FSR), fractional breakdown rate (FBR), and net protein balance (FSR - FBR) (NET) was examined in 8 healthy, previously unfit men and women [21.0+/- 0.4 y, 163.7+/- 4.4 cm, 75.6+/- 5.7 kg, 33.5+/- 4.1% body fat, VO(2 peak) 38.6+/- 2.3 mL/(kg.min)] fed eucaloric diets providing 0.85 g protein/(kg.d) for the 6-wk study. Measurements were made at baseline after 2 wk of diet intervention only, and after 4 wk of aerobic exercise training and diet intervention. Primed continuous infusions of ring-[(2)H(5)]-phenylalanine (2 micromol/kg; 0.05 micromol/(kg.min) and [(15)N]-phenylalanine (2 micromol/kg; 0.05 micromol/(kg.min) were used to assess skeletal muscle protein turnover at rest via the precursor-product method. Endurance training improved cardiovascular fitness, with a significant increase in VO(2 peak) (P<0.01) and a significant decrease in running time on a standard course (P<0.01). There were o significant changes in body mass or composition. There was a significant increase in FSR (0.077+/- 0.007 vs. 0.089+/- 0.006%/h, P<0.05) and decrease in NET (FSR - FBR) (-0.023 +/-0.004 vs. -0.072 +/- 0.012%/h, P < 0.05); FBR tended to increase (0.105+/- 0.014 vs. 0.143+/- 0.018%/h; P=0.06) after training. Findings show that aerobic training for 4 wk increases skeletal muscle protein turnover in previously unfit subjects.