Alterations in neonatal airway mechanics resulting from ventilatory therapies are implicated in airway collapse and chronic disease. Quantifying the functional impact of mechanical ventilation (MV) on the neonatal airway and elucidating the time course of these changes will support development of protective therapies. The objective of this study was to test the hypothesis that conventional MV would result in decreased static and dynamic elastance of an isolated tracheal segment and thinning of the muscle (trachealis) region of the tracheal wall in a time dependent manner. Tracheal segments were isolated in newborn lambs spontaneously breathing through the distal trachea; segments were MV (n = 7; PIP/PEEP = 35/5 cmH2O; 40 breaths/min) or instrumented, non-ventilated (SHAM; n = 7; PIP/PEEP = 0/0 cmH2O) for 4 hr. At baseline and hourly, tracheal segments were filled with saline, and static pressure-volume curves were constructed as the pressure response to stepwise volume infusions. Then, cross-sectional ultrasound images were captured at 0 cmH2O on SHAM, and at 0 cmH2O, peak inspiratory pressure (PIP) and positive end expiratory pressure (PEEP), on MV tracheae for subsequent dimensional analysis. Tracheal elasticity indices were derived from static pressure-volume data, and during dynamic ventilation using ultrasound images to calculate the stress-strain relationships. Over 4 hr of MV, tracheal internal diameter (ID) increased (14%; P < 0.05). Markers of tracheal mechanical properties indicated a decrease in elasticity under both static (bulk modulus; 28%; P < 0.05) and dynamic (elastic modulus; 282%; P < 0.05) conditions, indicating a significant alteration in elastic components. No time dependent changes were identified in dimensions or mechanical properties in the SHAM group. Conclusions: MV results in dimensional alterations that increased anatomical dead space and reduced static and dynamic elastance of the neonatal trachea.