BACKGROUND AND AIM: Carbon dioxide (CO2) insufflation devices are commonly used for endoscopic examination and treatment. In this prospective randomized controlled trial (RCT), we compared patient acceptance, cardiovascular tolerance,and autonomic nervous responses between patients receiving air insufflation and CO2 insufflation. METHODS: We initially enrolled 170 patients and, of these, 158 patients in total were analyzed (air group, 83; CO2 group, 75). Autonomic nervous responses were evaluated by analysis of heart rate variability (HRV). Primary end point was superiority in the effects of CO2 insufflation on the autonomic nervous system by HRV analysis. RESULTS: Visual analog scale disclosed significantly less abdominal pain and abdominal fullness with CO2. Percentage heart rate change rate at 1 h and 4 h after the procedure was also significantly lower in the CO2 group than in the air group (1 h after: P < 0.01, 4 h after: P < 0.05). Comparison based on age showed that % heart rate change was significantly lower in the younger CO2 patients (just after colonoscopy and 1 h after: P < 0.01, 4 h after: P < 0.05), but this difference was not apparent in an older group of patients. CONCLUSIONS: This is the first RCT showing that colorectal polypectomy using CO2 insufflation significantly decreases abdominal pain and abdominal fullness common in such patients with lowered stress to the autonomous nervous system. The effects using CO2 insufflation on the sympathetic nervous system also seemed to be more prominent among younger patients.