To investigate whether free melatonin may be better suited to reveal age-related changes, we studied the circadian rhythm alterations in saliva melatonin levels during aging. Special attention was paid to the question as to how the free melatonin rhythms change in aging and when such changes take place. A total of 52 healthy volunteers participated in the study consisting of young, middle-aged, old and the oldest groups. In each subject, a total of 12 time-point salivary melatonin samples was taken over 24 hr. Of the 52 data sets, 51 exhibited significant circadian rhythm over 24 hr by using the base cosine function analysis to fit the data. A clear circadian rhythm of salivary melatonin was present in all age groups. The decline in nocturnal peak levels (amplitude) in salivary melatonin was found in old and the oldest subjects. Both the old and the oldest subjects showed an increased daytime (baseline) melatonin levels. The off-set melatonin levels were more than two times higher in the oldest group than that in the other groups indicating a delayed phase of salivary melatonin. Most strikingly, we found that a step-wise decrease in the circadian rhythms of saliva melatonin occurred early in life, around 40 yr of ages. The middle-aged subjects had only 60% of the amplitude of the young subjects. In addition, the middle-aged subjects showed the longest peak levels duration and the lowest daytime melatonin levels. The present study showed that the alterations in the circadian rhythms of salivary melatonin begin during middle-age. Our results showed that salivary melatonin measurement is a reliable, sensitive and easy method to monitor changes in the circadian rhythms of melatonin during the course of aging.