The plasma free amino acid (PFAA) profile is affected by various nutritional conditions, such as the dietary energy balance. Regarding the clinical use of PFAA profiling, it is of concern that differences in food ingestion patterns may generate systematic errors in a plasma amino acid profile and constitute a confounding factor in assessment. In this study, the overnight impact of the dietary energy balance on the postprandial plasma amino acid profile was investigated to elucidate in particular the effects of high protein meals typical in Japanese cuisine. We conducted diet-controlled, crossover trials in eleven healthy male volunteers aged 40–61 y. They consumed either a normal meal (meal N) or high protein meal (meal H) at dinner. Forearm venous blood was collected, and plasma amino acid concentrations were measured before dinner and the next morning. We found that a high protein meal in the evening that contained 40% energy would significantly increase the PFAA concentration the next morning, even more than 12 hours after the meal. Among amino acids, the most significant difference was observed in the branched-chain amino acids (BCAAs) and in some urea-cycle related compounds. If the subject consumed the high protein diet at dinner, the PFAA profile after overnight fasting might be still affected by the meal even 12 hours after the meal, suggesting that the PFAA profile does not reflect the subject's health condition, but rather the acute effect of high protein ingestion.