Background: Functional overactive bladder disorder is one of the most prevalent presentations of bladder and bowel dysfunction in children, and it is associated with lower overall cardiac autonomic and parasympathetic activity. Antimuscarinics are the most frequently used pharmacological agents for treatment of children with functional overactive bladder disorder; however, there is a gap in the literature in describing the effect of antimuscarinics on the autonomic profile of this population. Objective: The aim of the study was to assess the cardiac parasympathetic activity before and after 12 weeks of oxybutynin treatment in children with overactive bladder. Methods: This was a single-institution prospective cohort study. Cardiac autonomic activity was assessed during storage and voiding phases of the bladder function via spectral analysis of heart rate variability and impedance cardiography. The primary outcome measure was high frequency, a proxy for parasympathetic nervous system activity. Parameters of uroflow study, severity of symptoms, and quality of life outcomes were also assessed. Results: Ten children (7 females) diagnosed with overactive bladder with a median age of 10 years (range = 6–14) were followed up for a median treatment duration of 11.8 weeks (range = 6–19.4). After treatment, there was a significant reduction in high frequency during the storage phase (median change = −24.17%, p = 0.047). No change was observed in the other outcome measures except for the overall Symptom Score for Dysfunctional Elimination Syndrome after treatment (5-point decrease, p = 0.034) (Summary Table). Discussion: The findings of the present follow-up study suggest that the use of oxybutynin in children with overactive bladder is associated with a significant reduction in the activity of the parasympathetic nervous system. The clinical implications of this finding are important because similar autonomic profiles (as markers of chronic stress) have been shown to be associated with increased inflammation and are found in major chronic diseases. The authors caution making a clinical connection between the heart rate variability profile of the patients in this study and patients with chronic diseases because oxybutynin is usually not administered as long-term treatment for overactive bladder. Conclusion: Use of oxybutynin was associated with reduction in cardiac parasympathetic activity of children with functional overactive bladder. Further investigation into the role of the autonomic nervous system as a treatment target in the management of these children is warranted. [Table presented] © 2019 Journal of Pediatric Urology Company