The purpose of this study was to explore the effect of massage on the urine proteome of healthy people. In this study, subjects underwent 1h whole body massage. Urine samples were collected at 0, 2, and 24h after the massage and urine proteins were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Compared with the control (before massage), 41 differential proteins were identified in the group 2h after massage and 29 in the group 24h after massage. The biological process of protein enrichment in the 2h group after the massage was catecholamine biosynthesis, which is related to the promotion of metabolism and the regulation of neural activities, while the differentially expressed proteins in the 24h group after massage were not involved in any effective biological process, indicating that the difference in urine proteome caused by massage gradually decreased after 24h. The results suggest that the proteome of urine can reflect changes in the body after minor massage stimuli, providing a new way to study the efficacy of massage therapy.