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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 4-day ( days 1–4) baseline period of a controlled energy balanced diet while maintaining their normal physical activity level followed by 7 days ( days 5–11) of a 1,000 kcal/day increase in physical activity above their normal activity levels. Two subject groups, one sedentary (Sed, mean V̇o2peak: 39 ml·kg−1·min−1, n = 7) and one fit (FIT1, mean V̇o2peak: 56 ml·kg−1·min−1, n = 8) increased energy intake to maintain energy balance throughout the 7-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/day exercise-induced energy deficit. Of these, FIT2 received an adequate protein diet (0.9 g/kg), and FIT3 received a high-protein diet (1.8 g/kg). For all four groups, IGF-I, IGFBP-3, and the acid labile subunit (ALS) were significantly decreased by day 11 (27 ± 4%, 10 ± 2%, and 19 ± 4%, respectively) and IGFBP-2 significantly increased by 49 ± 21% following day 3. 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.