Brain serotonin influences food intake and mood. It is synthesised from tryptophan (Trp) of which uptake in the brain is dependent on plasma ratio of tryptophan to the sum of other large neutral amino acids (Trp-LNAA). A carbohydrate-rich diet increases this ratio, whereas a protein-rich diet decreases it. Yet, if the protein source is α-lactalbumin the ratio increases. It is, however, unknown whether this also happens in the context of a regular diet (15% protein). We studied the effect of an α-lactalbumin supplement combined with regular diet on plasma Trp-LNAA ratio, serum prolactin (marker of serotonin synthesis), food intake, appetite, macronutrient preference and mood. Eighteen healthy males participated in a double-blind, randomised, placebo-controlled, crossover study. One hour after breakfast they received a drink containing α-lactalbumin and carbohydrates (AS) or carbohydrates (PS) only. Plasma Trp-LNAA ratio, serum prolactin, food intake, appetite, macronutrient preference and mood were assessed before and 90 min after consumption of the supplement. Changes of plasma Trp-LNAA ratio differed (P<.001) between both supplements, increasing by 16% after AS and decreasing by 17% after PS. Decrease of serum prolactin was slightly smaller after AS than after PS (P=.083). Appetite, food intake, macronutrient preference or mood did not differ between supplements. We conclude that an α-lactalbumin-enriched supplement combined with a regular diet increases plasma Trp-LNAA ratio and may influence serum prolactin, but we could not demonstrate effects on appetite, food intake, macronutrient preference and mood.