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In the context of Mediterranean aquaculture little attention has been paid to the manipulation of feeding frequency at the on-growing phase. The effects of different feeding frequencies: one meal per day, two meals per day, three meals per day on growth, digestive enzyme activity, feed digestibility and plasma biochemistry were studied in gilthead sea bream (Sparus aurata, L. 1758) fed with high and low fishmeal and fish oil levels. Isonitrogenous and isolipidic extruded diets were fed to triplicate fish groups by a fixed ration over 109 days. No significant effects of feeding frequency on overall performance, feed efficiency and feed digestibility during the on-growing of gilthead sea bream fed high or low fishmeal and fish oil dietary level were observed. Pepsin activity showed an apparent decrease in fish receiving more than one meal a day which was not compensated by an increased production of alkaline proteases particularly in fish fed on low FM. Although there were no effects on growth and feed utilisation at increasing feeding frequency, trypsin decreased significantly with an increasing number of meals only under low FMFO diet. Thus, it seemed that consecutive meals could have amplified the potential trypsin inhibitor effect of the vegetable meal-based diet adopted. Results of the plasma parameters related to nutritional and physiological conditions were not affected by feeding frequency. The higher level of plasma creatinine detected in fish fed a single daily meal with high FMFO level seems to be within physiological values in relation to the higher protein efficiency observed with this diet. According to the results, gilthead sea bream seems able to maximise feed utilisation regardless of the number of meals, and this could be a useful indicator for planning feeding activity at farm level to optimise growth of fish and costs of feeding procedures.