Many passengers experience discomfort during flight because of the effect of low humidity on the skin, eyes, throat, and nose. In this physiological study, we have investigated whether flight and low humidity also affect the tympanic membrane. From previous studies, a decrease in admittance of the tympanic membrane through drying might be expected to affect the buffering capacity of the middle ear and to disrupt automatic pressure regulation. This investigation involved an observational study onboard an aircraft combined with experiments in an environmental chamber, where the humidity could be controlled but could not be made to be as low as during flight. For the flight study, there was a linear relationship between the peak compensated static admittance of the tympanic membrane and relative humidity with a constant of proportionality of 0.00315 mmho/% relative humidity. The low humidity at cruise altitude (minimum 22.7 %) was associated with a mean decrease in admittance of about 20 % compared with measures in the airport. From the chamber study, we further found that a mean decrease in relative humidity of 23.4 % led to a significant decrease in mean admittance by 0.11 mmho [F(1,8)=18.95, P= 0.002], a decrease of 9.4 %. The order of magnitude for the effect of humidity was similar for the flight and environmental chamber studies. We conclude that admittance changes during flight were likely to have been caused by the low humidity in the aircraft cabin and that these changes may affect the automatic pressure regulation of the middle ear during descent.