Obesity associated with a sedentary lifestyle can lead to changes in the immune system balance resulting in the development of inflammatory diseases. The aim of this study was to compare lymphocyte activation mechanisms between overweight children practicing regular circus physical exercises with non-exercised children. The study comprised 60 pubescent children randomly divided into 4 groups: Overweight Children (OWC) (10.67 plus or minus 0.22 years old), Overweight Exercised Children (OWE) (10.00 plus or minus 0.41 years old), Eutrophic Children (EC) (11.00 plus or minus 0.29 years old) and Eutrophic Exercised Children (EE) (10.60 plus or minus 0.29 years old). OWE and EE groups practiced circus activities twice a week, for 4.3 plus or minus 0.5 and 4.4 plus or minus 0.5 months, respectively. Percentage of T regulatory cells (Treg) and the expression of CD95 and CD25 in CD4+ lymphocytes were evaluated by flow cytometry. Lymphocyte proliferation capacity was measured by 14C-thymidine incorporation and mRNA expression of IL-35, TGF-beta, IL-2 and IL-10 by real-time PCR. Lymphocyte proliferation was higher in OWC and OWE groups compared with the EC (3509 plus or minus 887; 2694 plus or minus 560, and 1768 plus or minus 208 cpm, respectively) and EE (2313 plus or minus 111 cpm) groups. CD95 expression on lymphocytes was augmented in the EC (953.9 plus or minus 101.2) and EE groups (736.7 plus or minus 194.6) compared with the OWC (522.1 plus or minus 125.2) and OWE groups (551.6 plus or minus 144.5). CTLA-4 expression was also lower in the OWC and OWE groups compared with the EC and EE groups. Percentage of Treg, IL-35, and IL-10 mRNA expression were lower in the OWC and OWE groups compared with the EC and EE groups. In conclusion, overweight children present altered immune system balance characterized by elevated lymphocyte proliferation due to a decrease in T regulatory cell percentage. These effects were partially reverted by moderate physical exercise, as demonstrated by decreased lymphocyte proliferation.