Due to the increasing prevalence of obesity among children, Shuttle Run Test (SRT) has been used as primary outcome for assessment of both physical performance and responses to different physical training programs. Thus, this study aimed to compare the performance on SRT between obese and non-obese children and the reproducibility of two SRTs carried out on different days.A cross-sectional study in which 40 children, aged from 8 to 10, were recruited from a public school. This study consisted of three visits in each school. On the first visit, we carried out a medical screening for recruited children. On the second visit, we applied the first SRT (SRT1), which was repeated on the third visit (SRT2, 24 hours apart).There was a significant difference in the distance traveled by non-obese in comparison with obese children (mean difference: 88 meters and 95% of confidence interval: 21 meters to 156 meters). Time and distance traveled of 27 children were higher in the SRT1, whereas nine children traveled a greater distance and presented higher testing time on the SRT2, with only four children showing the same distance traveled in both tests. Although both groups presented with reduction from the SRT1 to SRT2, this reduction was not significant (non-obese: 342 ± 97 meters to 319 ± 106 meters, respectively; obese: 269 ± 91 meters to 246 ± 90 meters, respectively). In obese children, the distance traveled in the best SRT had correlation with weight (r = -0.495, p = 0.043) and BMI (r = - 0.602, p = 0.011). No correlation was observed in the non-obese children.Overweight children had lower performance in SRT. Although reproducible, the best performance was in the first test, which leads us to suggest applying only one test.