||Rationale: Virtual bronchoscopic navigation (VBN) guidance to peripheral pulmonary lesions is often limited by insufficient segmentation of the peripheral airways. Objectives: To test the effect of applying positive airway pressure (PAP) during CT acquisition to improve segmentation, particularly at end-expiration. Methods: CT acquisitions in inspiration and expiration with 4 PAP protocols were recorded prospectively and compared to baseline inspiratory acquisitions in 20 patients. The 4 protocols explored differences between devices (flow vs. turbine), exposures (within seconds vs. 15-min) and pressure levels (10 vs. 14 cmH2O). Segmentation quality was evaluated with the number of airways and number of endpoints reached. A generalized mixed-effects model explored the estimated effect of each protocol. Measurements and Main Results: Patient characteristics and lung function did not significantly differ between protocols. Compared to baseline inspiratory acquisitions, expiratory acquisitions after 15 min of 14 cmH2O PAP segmented 1.63-fold more airways (95% CI 1.07–2.48; p = 0.018) and reached 1.34-fold more endpoints (95% CI 1.08–1.66; p = 0.004). Inspiratory acquisitions performed immediately under 10 cmH2O PAP reached 1.20-fold (95% CI 1.09–1.33; p < 0.001) more endpoints; after 15 min the increase was 1.14-fold (95% CI 1.05–1.24; p < 0.001). Conclusions: CT acquisitions with PAP segment more airways and reach more endpoints than baseline inspiratory acquisitions. The improvement is particularly evident at end-expiration after 15 min of 14 cmH2O PAP. Further studies must confirm that the improvement increases diagnostic yield when using VBN to evaluate peripheral pulmonary lesions.