This study evaluated gastrointestinal emptying, enzyme activity and daily rhythmic patterns of protein digestion in different portions of the gastrointestinal tract of Nile tilapia (Oreochromis niloticus). Thirty-six juveniles were distributed in six tanks (100-L each) kept in a recirculating water system and fed twice a day for 30 days. The fish were then fed at ZT1 h (Zeitgeber time = ZT) and sampled at six times: ZT1.5, ZT5, ZT9, ZT13, ZT17 and ZT21 h (belonging to 0.5, 4, 8, 12, 16 and 20 h, post-feeding, respectively). The parameters evaluated were stomach weight, presence of food in the stomach, passage of food in the intestine, acid protease activity in the stomach and trypsin and chymotrypsin activity in the different portions of the intestine. The greatest stomach weight was found right after feeding in ZT1.5 h. The presence of food in the stomach and intestine showed significant variations among sampling points. All enzymes displayed daily variations in their activity in all gut portions analyzed, except for trypsin in the intermediate portion. Acid protease activity was higher at ZT5 h, and the acrophase of the daily variation was located at ZT06:24 h. Trypsin activity differed significantly among sampling times in the initial and final intestine and the acrophases were located at ZT3:35 and ZT12:05 h, respectively. Chymotrypsin activity differed significantly among sampling times in all portions and acrophases appeared sequentially at ZT03:45, ZT7:01 and ZT10:30 h, in the initial, intermediate and final intestine, respectively. In conclusion, the passage of food in the gastrointestinal tract influences protein hydrolysis and correlates with proteolytic enzyme activity over 20 h. Furthermore, there is a rhythmicity in the activities of acid protease, trypsin and chymotrypsin, with acrophases located through the light phase.