Purpose: The aim of this study was to determine the influence of emotional expression in spectral energy distribution in professional theater actors. Study Design: The study design is a quasi-experimental study. Method: Thirty-seven actors, native Spanish speakers, were included. All subjects had at least 3 years of professional experience as a theater actor and no history of vocal pathology for the last 5 years. Participants were recorded during a read-aloud task of a 230-word passage, expressing six different emotions (happiness, sadness, fear, anger, tenderness, and eroticism) and without emotion (neutral state). Acoustical analysis with long-term average spectrum included three variables: the energy level difference between the F1 and fundamental frequency (F0) regions, ratio between 1-5 kHz and 5-8 kHz, and alpha ratio. Results: All the different emotions differ significantly from the neutral state for alpha ratio and 1-5/5-8 kHz ratio. Only significant differences between "joy," "anger," and " eroticism" were found for L1-L0 ratio. Statistically significant differences between genders for the three acoustical variables were also found. Conclusion: The expression of emotion impacts the spectral energy distribution. On the one hand emotional states characterized by a breathy voice quality such as tenderness, sadness, and eroticism present a low harmonic energy above 1 kHz, high glottal noise energy, and more energy on F0 than overtones. On the other hand, emotional states such as joy, anger, and fear are characterized by high harmonic energy greater than 1 kHz (less steep spectral slope declination), low glottal noise energy, and more energy on the F1 than F0 region.