Introduction: Obesity is one of the most prevalent chronic diseases in childhood, being an important public health issue. Excessive weight has been associated with autonomic dysfunction but the evidence in children is scarce. Therefore, the aim of this study was to assess the effect of overweight and obesity on the autonomic nervous system activity, in children.Material and Methods: Data from a cross-sectional study of 1602 children, aged 7 to 12 years, was used and 858 children were included in the analysis. Body mass index was calculated and classified according to criteria of the World Health Organization (WHO), Centers for Disease Control and Prevention (CDC) and the International Obesity Task Force (IOTF). Body composition was characterized by bioelectrical impedance. Linear regression models were used to determine the association between body mass index, body composition and the autonomic nervous system activity, assessed by pupillometry.Results: Average dilation velocity was higher among children with obesity, according to the CDC and percentage of body fat criteria (β = 0.053, 95% CI = 0.005 to 0.101 and β = 0.063, 95% CI = 0.016 to 0.109, respectively). The same trend was observed for WHO and IOTF criteria (β = 0.045, 95% CI = -0.001 to 0.091, and β = 0.055, 95% CI = -0.001 to 0.111, respectively). CDC and WHO body mass index z-scores were also positively associated with the values of average dilation velocity (rs = 0.030, p = 0.048; and rs = 0.027, p = 0.042, respectively).Conclusion: Our findings suggest an association between body mass and changes in the autonomic activity, Moreover, this study provides proof of concept for interventions targeting the prevention/treatment of obesity in children that may offer some benefit in re-establishing the balance of the autonomic nervous system, and subsequently preventing the consequences associated with the autonomic nervous system dysfunction.