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In contrast to humans, a tryptophan (TRP)-free amino acid (AA) mixture only leads to moderate depletion in plasma TRP levels in adult rats. In this study we evaluated the effects of an acute administration of a TRP-free protein–carbohydrate nutritional mixture in adult male Wistar rats. Plasma amino acid levels were examined at 2 and 4 h starting after the first administration. Furthermore, the concentrations of amino acid, serotonin (5-HT), dopamine (DA) and their metabolite (5-hydroxyindolacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC), respectively) were measured within the striatum, hippocampus and cortex. In the TRP depleted animals, the TRP/ΣLNAA ratio (LNAA: large neutral amino acids) was substantial decreased at 2 and 4 h after the first administration of the oral administration (by 71 and 78%, respectively). Four hours after treatment central TRP and 5-HT concentrations were decreased by 50%. Both peripheral and central TRP levels returned to basal values in the group treated with the nutritional mixture supplemented with TRP. Surprisingly, tyrosine levels were also reduced after oral administration of the protein–carbohydrate mixture without affecting central DA concentrations. In conclusion, the TRP-free protein–carbohydrate nutritional mixture appears to be an efficient tool to substantially reduce plasma and central TRP levels in adult rat.