Tryptophan (TRP) supplemented diets have been shown to have therapeutic effects in farmed animals including fish by modulating the activity of the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT). The effects reported in fish have been obtained using individually-housed fish and include a reduction in stress response, aggression and stress-induced anorexia. In land farmed animals, TRP supplemented diets have also been shown to improve meat quality as a result of reduced stress during slaughter while in fish no data is currently available. This study aims at investigating whether short-term supplementation with TRP supplemented diets changes brain serotonergic activity and the stress response associated with slaugh-ter handling in grouped-housed Nile tilapia Oreochromis niloticus. Adult fish (n = 108, 490.6 ± 4.0 g, 12 individuals per tank) were exposed to one of the three treatments (triplicates per treatment were used): con-trol (0.48 g/100 g), TRP 4× (1.87 g/100 g) and TRP 10× (4.45 g/100 g) diets during 7 days. Afterwards, half of the fish in each tank were subjected to an acute stressor consisting of a combination of crowding and chas-ing, just prior to slaughter. The other half of the fish represented undisturbed conditions. Blood and brain samples were collected for cortisol and serotonergic activity analyses, respectively. Flesh quality was also assessed in both undisturbed and stressed fish for all treatments by measuring muscle pH and rigor mortis over a 72 h period. Results showed that the highest TRP supplemented diet (TRP 10×) induced a significant reduction in undisturbed plasma cortisol (10.57 ± 2.71 ng/ml) as compared to TRP 4× (24.93 ± 3.19 ng/ml) and control diets (18.69 ± 2.94 ng/ml) and no effect on post-stress cortisol levels. After stress, the major 5-HT metabolite (5-hydroxyindoleacetic acid, 5-HIAA) was higher in the TRP 10× (471.31 ± 60.95 ng/g) as com-pared to the other diets (TRP 4×: 313.52 ± 30.12 ng/g; control: 260.36 ± 19.65 ng/g). Stress before slaughter induced a significant increase in plasma cortisol (from 18.40 ± 1.76 ng/ml under undisturbed conditions to 80.34 ± 7.16 ng/ml), however, it was not sufficient to cause a faster deterioration of flesh quality. TRP supple-ment diets had also no effect on muscle pH and rigor mortis during the 72 h observation period. In conclusion, this study showed that only the highest levels of supplementation (10× the control diet) affect serotonergic activity. However, these levels did not result in reduced stress responsiveness or improved flesh quality when an acute stressor is applied before slaughter. Therefore, these results underline the fact that effects of TRP on cortisol production are dose-and context-dependent, and further experiments are needed to determine under which conditions the optimal effect is obtained.