Background: A sudden increase in exercise and energy expenditure is associated with an increase in protein turnover and nitrogen excretion. This study examined how a sudden increase in exercise-induced energy expenditure affected whole body protein metabolism and nitrogen balance in people of differing levels of aerobic fitness. We hypothesized that alterations in whole-body protein turnover would be attenuated, and nitrogen balance would be preserved, in individual with higher levels of aerobic fitness., Methods: Eleven men, categorized as either having a lower (LOW-FIT; n = 5) or higher (FIT; n = 6) aerobic fitness level, completed a 4-d baseline period (BL) of an energy balance diet while maintaining usual physical activity level, followed by a 7-d intervention consisting of 1,000 kcal·d⁻¹ increased energy expenditure via exercise (50-65% VO₂peak). All volunteers consumed 0.9 g protein.kg⁻¹·d⁻¹ and total energy intake was adjusted to maintain energy balance throughout the 11-d study. Mean nitrogen balance (NBAL) was determined for BL, days 5-8 (EX1), and days 9-11 (EX2). Whole-body protein turnover was derived from phenylalanine and tyrosine kinetics assessed while fasting at rest on days 4, 7, and 12 using a priming dose of L-[ring-¹⁵N]tyrosine and a 4-h primed, continuous infusion of L-[¹⁵N]phenylalanine and L-[ring-²H₄]tyrosine., Results: A significant main effect of time indicated that NBAL increased over the course of the intervention; however, a group-by-time interaction was not observed. Although FIT demonstrated a lower net protein oxidation and higher net protein balance compared to LOW-FIT, neither the effect of time nor a group-by-time interaction was significant for Phe flux, net protein oxidation, or derived whole-body protein synthesis and net protein balance., Conclusion: The absence of significant group-by-time interactions in protein metabolism (i.e., NBAL and whole-body protein turnover) between LOW-FIT and FIT males suggest that aerobic fitness level does not modulate protein "sparing" in response to an unaccustomed increase in energy expenditure.