L-theanine is one of the major amino acids, predominantly found in green tea leaves. Historically, this amino acid has been known as a relaxing agent. The current study examined the effects of L-theanine and a placebo on the automatic nervous system during sleep, using the pulse frequency demolition method. For this purpose, a cross over double blind study was performed on 20 females who were in their postmenopausal period and of 57.3±3.9 years old. During the study, each participant ingested 200mg of L-theanine or placebo before sleeping, for six consecutive days. The results showed that L-theanine significantly inhibited the excitement of the sympathetic nervous system and tended to noticeable enhance the activity of the parasympathetic nervous system, during sleep. Thus, the physiological effect of L-theanine on the automatic nervous system as estimated, was found to play a role in improving the quality of sleep. This study suggested that L-theanine was useful for promoting comfortable living states.