OBJECTIVE: Listening to Mozart K.448 has been demonstrated to improve spatial task scores, leading to what is known as the Mozart Effect. However, most of these reports only describe the phenomena but lack the scientific evidence needed to properly investigate the mechanism of Mozart Effect. In this study, we used electroencephalography (EEG) and heart rate variability (HRV) to evaluate the effects of Mozart K.448 on healthy volunteers to explore Mozart Effect. DESIGN: An EEG-based post-intervention analysis. SETTING: Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. PARTICIPANTS: Twenty-nine college students were enrolled. They received EEG and electrocardiogram examinations simultaneously before, during and after listening to the first movement of Mozart K.448. MAIN OUTCOME MEASURE: EEG alpha, theta and beta power and HRV were compared in each stage. RESULTS: The results showed a significant decrease in alpha, theta and beta power when they listened to Mozart K.448. In addition, the average root mean square successive difference, the proportion derived by dividing NN50 by the total number of NN intervals, standard deviations of NN intervals and standard deviations of differences between adjacent NN intervals showed a significant decrease, while the high frequency revealed a significant decrease with a significantly elevated low-frequency/high-frequency ratio. CONCLUSION: Listening to Mozart K.448 significantly decreased EEG alpha, theta and beta power and HRV. This study indicates that there is brain cortical function and sympathetic tone activation in healthy adults when listening to Mozart K.448, which may play an important role in the mechanism of Mozart Effect.