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Prolonged voluntary abdominal breathing (VAB) is considered a basic component of Zen meditation.We previously demonstrated that prolonged VAB using visual feedback increased EEG alpha activity and improved mood.To gain insight into the neurophysiological mechanisms, we investigated hemodynamic changes in the prefrontal cortex (PFC) in 15 healthy volunteers, using near-infrared spectroscopy during a 20-minute session of VAB.In addition, we assessed mood changes using the profile of mood states (POMS) questionnaire and examined electroencephalography (EEG) changes during the VAB session.Using a high-performance liquid chromatography(HPLC) system, we determined the serotonin (5-HT) levels in whole blood to evaluate the augmentation of 5-HT release in the brain.We found that the level of oxygenated hemoglobin in the anterior PFC was significantly increased during VAB, accompanied by a reduction in feelings of negative mood.Activation of the anterior PFC may contribute to this mood improvement because anterior PFC tends to be activated by task involving emotion.EEG analysis revealed increased alpha band activity and decreased theta band activity during and after VAB.EEG changes were correlated with a significant increase in whole blood 5-HT levels.Because brain 5-HT is known to inhibit cholinergic neurons projecting to broad cortical areas, we propose that the augmented brain 5-HT release may induce cortical attenuation, resulting in the emergence of alpha band activity.These results suggested that activation of the anterior PFC and 5-HT system may be responsible for improvement of negative mood and EEG signal changes during VAB.