Introduction: Prolonged intraluminal and intraperitoneal insufflation with gas is a critical component of natural orifice transluminal endoscopic surgery (NOTES); however, the physiology of serial combined insufflation has not been evaluated. This study describes the physiologic effects of prolonged visceral followed by intraperitoneal insufflation with room air.Methods: Five swines underwent sequential insufflation of the stomach (60 min) and peritoneum (60 min) with room air at a pressure of 15 mmHg. Physiologic parameters were measured at 10 minute intervals and arterial blood gas measurements were taken at 0, 30, and 60 minutes during each insufflation period. Outcome variables included: heart rate (HR), mean arterial pressure (MAP), stroke volume (SV), cardiac output (CO), systemic vascular resistance (SVR), oxygen saturation (O2 sat), end-tidal carbon dioxide (EtCO2), peak airway pressures (PAP), pH, pO2, and pCO2. The mean change of each variable from baseline at each timepoint was compared using Student’s t-test. Results: Sequential intragastric and peritoneal insufflation did not produce any significant changes in HR, MAP, SV, SVR, or CO. Following 60 minutes of intragastric insufflation, the SaO2 decreased from 98.4% to 97.4% (p=0.034). This also produced a respiratory acidosis with pH decreasing from 7.43 to 7.38 (p=0.006) with increased ETCO2 (39.6 mmHg to 46.2 mmHg, p=0.004) and pCO2 (47.36 mmHg to 54.8 mmHg, p=0.005). Subsequent intraperitoneal insufflation resulted in worsening respiratory acidosis (pH 7.34, p=0.037) with further increases in pCO2 (63.6 mmHg, p=0.005) and ETCO2 (51.6 mmHg, p=0.026). Peritoneal insufflation also increased PAP from 18.2 mmHg to 21.6 mmHg (p=0.005), but SaO2 did not decrease further.Conclusion: The prolonged serial insufflation of the stomach and peritoneal cavity required for NOTES procedures are well tolerated without significant hemodynamic effects. These maneuvers do, however, impact pulmonary mechanics and acid-base status. During NOTES procedures, these effects should be carefully monitored and mitigated via careful ventilator management.