Objectives: Vibrato is considered one of the most essential characteristics of the classical singing voice. Vibrato can be acoustically described by the rate, extent, onset, and regularity. The aim of this study was to determine the influence of emotional expression on acoustic parameters of vibrato in classically trained singers. Study Design: A prospective cohort study was performed. Methods: Thirty healthy classical singers were recruited for this study, 29 singers met inclusion criteria. Inclusion criteria for this study were as follow: 1) no history of vocal pathology in the past year, 2) to have at least 5 years of classical singing training. Each subject was asked to sing the phrase "I Love You," while expressing four different emotions (tenderness, anger, happiness, and sadness) and without emotion (neutral state). The musical tonality of the phrase was adapted to each singer's vocal classification. Subjects were also recorded at three levels of loudness (pianissimo, mezzo forte, and fortissimo), while expressing each emotion. Acoustical analysis was performed during the vowel /o/ of the word "Love" to determine rate of vibrato, the extent of vibrato, and vibrato jitter. Results: Vibrato parameters did not vary significantly when different emotions were expressed. However, vibrato jitter and extent did vary significantly between different levels of loudness. Significant differences were also noted in both rate and extent of vibrato when compared between sexes. Conclusions: Expression of emotions does not affect the acoustic parameters of vibrato, although some parameters are affected by loudness and gender.