Abstract 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 two 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%, p < 0.001). Regardless of the ventilatory mode, mean RH significantly differed between the two heater‐humidifiers (89.6 ± 6.7% vs 78.7 ± 6.8%, p = 0.003). The in vivo study included 10 neonates (mean ± SD gestational age: 25.7 ± 0.9 weeks and birthweight: 624.4 ± 96.1 g). Mean RH during HFOV was significantly lower than during IPPV (74.6 ± 5.7% vs. 83.0 ± 6.7%, p = 0.004). Conclusion RH 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.