Tidal ventilation is essential in supporting the transition to air-breathing at birth, but excessive tidal volume (V) is an important factor in preterm lung injury. Few studies have assessed the impact of specific V levels on injury development. Here, we used a lamb model of preterm birth to investigate the role of different levels of V during positive pressure ventilation (PPV) in promoting aeration and initiating early lung injury pathways. V was delivered as ) 7 mL/kg throughout (V), ) begun at 3 mL/kg and increased to a final V of 7 mL/kg over 3 min (V), or ) commenced at 7 mL/kg, decreased to 3 mL/kg, and then returned to 7 mL/kg (V). V, inflating pressure, lung compliance, and aeration were similar in all groups from 4 min, as was postmortem histology and lung lavage protein concentration. However, transient decrease in V in the V group caused increased ventilation heterogeneity. Following TMT-based quantitative mass spectrometry proteomics, 1,610 proteins were identified in the lung. Threefold more proteins were significantly altered with V compared with V or V strategies. Gene set enrichment analysis identified V specific enrichment of immune and angiogenesis pathways and V enrichment of metabolic processes. Our finding of comparable lung physiology and volutrauma across V groups challenges the paradigm that there is a need to rapidly aerate the preterm lung at birth. Increased lung injury and ventilation heterogeneity were identified when initial V was suddenly decreased during respiratory support at birth, further supporting the benefit of a gentle V approach. There is little evidence to guide the best tidal volume (V) strategy at birth. In this study, comparable aeration, lung mechanics, and lung morphology were observed using static, incremental, and alternating V strategies. However, transient reduction in V was associated with ventilation heterogeneity and inflammation. Our results suggest that rapidly aerating the preterm lung may not be as clinically critical as previously thought, providing clinicians with reassurance that gently supporting the preterm lung maybe permissible at birth.T