Objectives: Emotions and stress affect voice production. There are only a few reports in the literature on how changes in the autonomic nervous system affect voice production. The aim of this study was to examine emotions and measure stress reactions during a voice examination procedure, particularly changes in the muscles surrounding the larynx. Material and Methods: The study material included 50 healthy volunteers (26 voice workers – opera singers, 24 control subjects), all without vocal complaints. All subjects had good voice quality in a perceptual assessment. The research procedure consisted of 4 parts: an ear, nose, and throat (ENT)‑phoniatric examination, surface electromyography, recording physiological indicators (heart rate and skin resistance) using a wearable wristband, and a psychological profile based on questionnaires. Results: The results of the study demonstrated that there was a relationship between positive and negative emotions and stress reactions related to the voice examination procedure, as well as to the tone of the vocal tract muscles. There were significant correlations between measures describing the intensity of experienced emotions and vocal tract muscle maximum amplitude of the cricothyroid (CT) and sternocleidomastoid (SCM) muscles during phonation and non-phonation tasks. Subjects experiencing eustress (favorable stress response) had increased amplitude of submandibular and CT at rest and phonation. Subjects with high levels of negative emotions, revealed positive correlations with SCM_max during the glissando. The perception of positive and negative emotions caused different responses not only in the vocal tract but also in the vegetative system. Correlations were found between emotions and physiological parameters, most markedly in heart rate variability. A higher incidence of extreme emotions was observed in the professional group. Conclusions: The activity of the vocal tract muscles depends on the type and intensity of the emotions and stress reactions. The perception of positive and negative emotions causes different responses in the vegetative system and the vocal tract. Int J Occup Med Environ Health. 2024;37(1):84–97