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Background: Inappropriate humidification of inspired gas during mechanical ventilation can impair lung development in extremely low birthweight (ELBW) infants. Humidification depends on multiple factors, such as the heater-humidifier device used, type of ventilation, and environmental factors. Few studies have examined inspired gas humidification in these infants, especially during high-frequency oscillatory ventilation (HFOV). Our objective was to compare humidity during HFOV and intermittent positive pressure ventilation (IPPV), in vitro and in vivo. Methods: In-vitro and in-vivo studies used the same ventilator during both HFOV and IPPV. The bench study used a neonatal test lung and 2 heater-humidifiers with their specific circuits; the in-vivo study prospectively included preterm infants born before 28 weeks of gestation. Results: On bench testing, mean absolute (AH) and relative (RH) humidity values were significantly lower during HFOV than IPPV (RH = 79.4% ± 8.1% vs 89.0% ± 6.2%, PConclusion: Relative humidity was significantly lower during HFOV than IPPV, both in vitro and in vivo. The type of heater-humidifier also influenced humidification. More systematic measurements of humidity of inspired gas, especially during HFOV, should be considered to optimize humidification and consequently lung protection in ELBW infants.