The purpose of this study was to determine the impact of dietary factors and exercise-associated factors on the response of IGF-I and its binding proteins (IGFBPs) during a period of increased physical activity. Twenty-nine men completed a four day (D1-D4) baseline period of a controlled energy balanced diet while maintaining their normal physical activity level followed by seven days (D5-D11) of a 1,000 kcal·d^-1 increase in physical activity above their normal activity levels. Two subject groups, one sedentary (SED, mean VO_2peak: 39 ml·kg^-1·min^-1, N=7) and one fit (FIT1, mean VO_2peak: 56 ml·kg^-1·min^-1, N=8) increased energy intake to maintain energy balance throughout the seven day intervention. In two other fit subject groups (FIT2, N=7 and FIT3, N=7), energy intake remained at baseline resulting in a 1,000 kcal·d^-1 exercise-induced energy deficit. Of these, FIT2 received an adequate protein diet (0.9 g·kg^-1), and FIT3 received a high protein diet (1.8 g·kg^-1). For all four groups, IGF-I, IGFBP-3, and the acid labile subunit (ALS) were significantly decreased by D11 (27±4%, 10±2%, and 19±4%, respectively) and IGFBP-2 significantly increased by 49±21% following D3. IGFBP-1 significantly increased only in the two negative energy balance groups, FIT2 (38±6%) and FIT3 (46±8%). Differences in initial fitness level and dietary protein intake did not alter the IGF-I system response to an acute increase in physical activity. Decreases in IGF-I were observed during a moderate increase in physical activity despite maintaining energy balance suggesting that currently unexplained exercise-associated mechanisms, such as increased energy flux, regulate IGF-I independent of energy deficit.