Rats trained to eat a single daily meal (MF rats), from 8:00-10:00 a.m., increased food intake from the 1st to the 12th (125%) day of feeding training. In this work we compared the influence of the higher food ingestion in the last meal and feeding training on hepatic gluconeogenesis. Thus, rats at the 1st (MF(1st day-5g) group) and 13th day (MF(13th day-5g) group) of training, refed with a fixed amount of food (5g) were employed. In addition, a third group of MF rats, refed on day 12 with 75% (12g) of the food ingested by MF rats on the 13th day of the feeding training (MF(13th day-12g)) was included. The experiments were performed at 22 h after meal (8:00 a.m.). Our results demonstrated that feeding training had a crucial role in determining gluconeogenesis from pyruvate (5 mM). Additionally, gluconeogenesis from L-glutamine (5 mM) was influenced by periodicity of eating and the amount of food ingested in the last meal. In contrast, gluconeogenesis from L-alanine (5 mM) was not influenced by both factors. In conclusion, our findings suggested that the hepatic gluconeogenesis was influenced by food ingestion and/or feeding training depending of the substrate investigated. These effects on gluconeogenesis may have implications for use in diabetic regimens.