Our aim was to study the alteration pattern and interaction of inflammatory tear cytokines during the course of a day. Using a prospective, experimental design, tears were collected from 28 healthy volunteers with normal eyes during the period from April 2004 to March 2005. Tears (10 microl) were collected by capillary outflow from each eye at 9:00, 12:00, 16:00, 21:00, and 24:00 h. The concentrations of inflammatory cytokines, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, and TNF-alpha were measured using cytometric bead arrays. Although the concentration of tear cytokines varied widely among eyes, the amount of cytokine had a specific alteration pattern in each eye during the course of a day. IL-1beta, IL-6, IL-10, IL-12p70, and TNF-alpha showed slight increases in the morning and the late evening. IL-8 remained low throughout the day. The alteration pattern of IL-8 was significantly different from those of TNF-alpha and IL-12p70 (P<0.01). The ratio of each pro-inflammatory cytokine to anti-inflammatory cytokine IL-10 did not significantly change throughout the day. The amount of tear cytokines changed during daytime with a specific pattern. This diurnal rhythm may influence symptoms of ocular surface diseases during the course of a day.