Contrary to carbohydrate and fat metabolism, the influence of a single exercise dose on protein metabolism has not been adequately explored yet. We assessed the effects of different exercise intensities and durations on blood protein changes and their association with carbohydrate (CHO) and fat metabolism in six eligible trained subjects. Subjects performed maximal incremental (IE100: at 100%VO2max) and submaximal continuous exercise (CE) at 75%VO2max for 30 min (CE75) and at 50%VO2max for 90 min (CE50). Blood samples were collected at rest (R), end of exercise (EE), and 1 h after recovery to assess blood urea nitrogen (BUN), plasma amino acids (AA), glucose, lactate, FFA, and glycerol. In IE100 blood lactate, CHO-oxidation (g/min), energy expenditure (kcal/min), and RER were significantly increased during rest (p < 0.05). CE50 induced significantly higher BUN, FFA, glycerol, and fat oxidation (g/min) (p < 0.05). At recovery, the mean sum of the free AA pool (µmol/L) reduced by 8% (p < 0.03) during CE50. Values for CE75 were between IE100 and CE50. Beside lipolysis, also proteolysis (BUN) was an important source of fuel for low-to-moderate intensity CE50. An increased uptake of AA from the plasma bed during CE50 suggests the importance for oxidation and synthesis of other metabolic sources such as gluconeogenesis necessary for recovery. Therefore, one needs to be cautious of protein diet following prolonged cycle exercise training.