Purpose. The purpose of our study was to test the prediction that if the tear film thins due to evaporation, rather than tangential flow, a high concentration of fluorescein in the tear film would show a greater reduction in fluorescent intensity compared to a low concentration of fluorescein due to self-quenching at high concentrations. Methods. Tear film thickness, thinning rate, and fluorescent intensity were measured continuously and simultaneously with a modified spectral interferometer in 30 healthy subjects with two different concentrations (2% followed by 10%) of 1 μL of liquid fluorescein on the eye. Measurements of fluorescein self-quenching (fluorescent efficiency as a function of fluorescein concentration) are described in an Appendix and are reported in arbitrary units. Results. Under low and high fluorescein concentration conditions, there were no differences in tear film thickness (P = 0.09) or thinning rates (P = 0.76). While the mean initial fluorescent intensity was similar between groups (637.47 ± 381.47 vs. 672.09 ± 649.72, P = 0.55), the mean rate of fluorescent decay was 4-fold faster in the high (16.57 ± 29.34) than in the low (4.11 ± 6.78) concentration group (P < 0.01). Conclusions. The large difference in the rate of fluorescent decay between groups can be explained by the effects of evaporation and self quenching of fluorescein; the latter is expected to be greater for high than for low fluorescein concentration. Fluorescence decay due to tangential flow would be expected to be similar at high and low fluorescein concentrations. This supports previous evidence that evaporation has the primary role in normal tear thinning between blinks. © 2012 The Association for Research in Vision and Ophthalmology, Inc.