PURPOSE. Tear evaporation should increase fluorescein concentration, causing fluorescence dimming from self-quenching for high but not low fluorescein concentration. This prediction was tested and compared to the predicted effect of "tangential flow" that fluorescence dimming should be similar for high and low concentrations. METHODS. A custom optical system was used for video recording of tear film fluorescence in 30 subjects. The subjects were asked to blink at the start of the recording and try to keep their eyes open for the rest of the 60-second recording. An initial recording was made after instillation of 1 lL 0.1% fluorescein followed by further recordings at 5-minute intervals using 0.5% and 5% fluorescein. RESULTS. Decay of fluorescence was considerably greater for the high (5%) concentration condition than for the low (0.1%) concentration. This is shown by "ratio images" (ratio of the intensity of a fluorescence image at a later time divided by that of an earlier image), fluorescence decay curves, fluorescence decay rates, and histograms of estimated tear thickness decrease. For example, for the high concentration condition, decay rates were higher than for the low concentration for all 30 subjects (P < 0.0001, binomial test). Additionally, breakup time was significantly reduced for the high compared to the low concentration condition. CONCLUSIONS. The greater fluorescence decay and more rapid breakup for the high concentration condition are the results expected if thinning and breakup are mainly due to evaporation, hence causing self-quenching. Fluorescence decay rate for the low concentration condition was not significantly greater than zero. © 2013 The Association for Research in Vision and Ophthalmology, Inc.