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INTRODUCTION: Sonotubometry allows an assessment of the Eustachian tube (ET) function under physiological conditions. The application of conventional sinus signals is not reliable enough for routine clinical use. The aim of this study was to investigate ET activity with so-called perfect sequences (PSEQ). PATIENTS AND METHODS: PSEQs generated by a custom-made device were applied in 25 healthy subjects. ET opening was induced by Toynbee manoeuvre, yawning, dry and water swallowing. All sonotubograms were qualitatively analysed according to their shape, and quantitatively according to increase of sound intensity, frequency of opening and opening duration. The sonotubometry results were combined with the patients' perception of tone variation. RESULTS: A number of 400 measurements were performed. In 92.75 % measurements the patients reported ET openings. These were confirmed by 81.5 % valid sonotubograms. The swallowing manoeuvres dry/water swallowing and Toynbee yielded with 94 %/90 % and 93 % valid measurements favourable results. Yawning was associated with 49 % valid measurements. Sound level increase was also significantly different in these manoeuvres when compared to yawning (p > 0.0001). Nasal decongestion had no influence on the results (p < 0.05). CONCLUSIONS: PSEQs allow an evaluation of ET function. The chosen manoeuvres trigger in healthy subjects objective ET openings with a high reliability. The application of PSEQs can detect ET activity with a high sensitivity and good specificity. By further technical refinements specificity may also be enhanced in future studies.