Background: The microscopically small middle ear structures require the otosurgeon to have a steady hand because instrument stability is a critical factor for a successful microsurgical procedure. Hand steadiness is mainly influenced by the tremor movements of the hand. The aim of this study was to measure hand tremor under simulated microsurgical conditions and to estimate the influence of different kinds of physical strain (e.g., physical exertion and hand exercise), as well as food abstinence and coffee consumption. Further, the effect of one-or two-handed manipulation and microsurgical experience was investigated. Methods: The hand movements of 16 adult subjects were assessed during a defined manual manipulation using a stapes model to simulate microsurgical procedures. A laserinterferometric-based displacement technique was developed to measure tremor amplitude and frequency, as well as maximum displacement, to evaluate the subjects' fine motor skills. Results: The mean tremor frequency across all measurements was 8.1 Hz and did not show any dependence on different kinds of physical strain. Two-handed manipulations showed significantly lower tremor amplitudes than one-handed performances. Tremor amplitude and maximum displacement did not change after hand exercise, food abstinence, and coffee consumption. However, after physical exertion, a significant increase in the tremor amplitude was found. Subjects with advanced microsurgical experience showed smaller tremor amplitudes for one-handed runs. Conclusion: The tremor data are interpreted as a recommendation to avoid physical exertion before microsurgery. In cases of absolute necessity for hand steadiness, two-handed manipulations are preferable. Further, hand steadiness might be improved by microsurgical training and experience.