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In our previous studies, the effects of effusion and pressure on sound transmission were investigated separately. The aim of this study is to investigate the combined effect of fluid and pressure on middle ear function. An otitis media with effusion model was created by injecting saline solution and air pressure simultaneously into the middle ear of human temporal bones. Tympanic membrane displacement in response to 90 dB SPL sound input was measured by a laser vibrometer and the compliance of the middle ear was measured by a tympanometer. The movement of the tympanic membrane at the umbo was reduced up to 17 dB by the combination of fluid and pressure in the middle ear over the auditory frequency range. The fluid and pressure effects on the umbo movement in the fluid–pressure combination are not additive. The combined effect of fluid and pressure on the umbo movement is different compared with that of only fluid or pressure change in the middle ear. Negative pressure in fluid–pressure combination had more effect on middle ear function than positive pressure. Tympanometry can detect the middle ear pressure of the fluid–pressure combination. This study provides quantitative information for analysis of the combined effect of fluid and pressure on tympanic membrane movement.