The aim of this study was to investigate whether a middle ear (ME) pressure rise noted during sleep is an effect of the recumbent position, the state of sleep or Eustachian tube (ET) function, respectively. Eleven subjects with no history of ear disease were subjected to continuous, 24-h, direct ME pressure measurements and tubal function tests, respectively. At the start of the measurements the subjects were in the erect position. This position was maintained for at least 2 h (during which they were permitted to conduct normal everyday activities). The subjects then rested in the recumbent position for at least 2 h, after which they again resumed the erect position with normal activities for a period of at least 2 h. Then the subjects went to sleep in the recumbent position. A mean pressure rise in the ME of 36.4 daPa was seen during sleep compared with being awake in the same position. The rise was not caused by tubal opening and active insufflation of air via the ET. The number of tubal openings was significantly fewer during sleep compared with resting awake and the erect position, respectively. The ME pressure was not significantly different in the erect position compared with the recumbent position while resting awake. Tubal function tests demonstrated results in accordance with normal tubal function for all subjects. In conclusion, the state of sleep induced a ME pressure rise, not the recumbent position per se. These findings support the significance of a two-directional gas diffusion for regulation of the ME pressure.