Aims: To know the effect of chronic commercial sweeteners consumption in lymphocytes of Peyer’s patches. Study Design: A prospective, longitudinal, comparative and experimental study. Place and Duration of Study: The study was conducted in the Nutrition Research Laboratory of the Faculty of Medicine of Universidad Autónoma del Estado de México (UAEMéx) between August 2018 and May 2019 and was approved by the Bioethics Committee. Materials and Methods: Two groups of male mice of different strains were used: 1) Balb/c and 2) CD1, both at 8 weeks-old age. The groups were divided into 4 subgroups: 1) Control (without sweetener), 2) Sucrose (table sugar, 41.66 mg/mL), and two groups of commercial sweeteners 3) Splenda® (sucralose 1.2%, with a concentration of 4.16 mg/mL), and 4) Svetia® (Steviol glycoside 0.025 g with a concentration of 4.16 mg/mL). The mice consumed the supplementation for 6 weeks. Also, were quantified plasma glucose, percentage of lymphocytes from Peyer’s patches, water and food consumption weekly. Results: Mice increased their body weight after 6 weeks of treatment. The animals of Control and Sucrose subgroups showed a significant body weight gain of 5 g compared with the Splenda® and Svetia® subgroups, which increased only 4 g. In the subgroup treated with Splenda®, the blood glucose was reduced significantly. Svetia® and Control groups consumed more water without sweetener. The differences in food consumption were between the subgroups, not between the strains. By the end, the percentage of lymphocytes from Peyer´s patches increased in the Sucrose subgroup but decreased significantly in other subgroups. Conclusion: The consumption of sweeteners may modify the lymphocyte population of Peyer's patches in the small intestine and this variation depends on the frequency of consumption the strain of the rodents and the type of sweetener.