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The purpose of this study is to compare visual versus software detection of non-invasive tear break-up with the KOWA DR-1α tear interferometer and investigate the relationship between non-invasive tear break-up time (NIBUT) and dry eye clinical severity. Tear interferometry with the KOWA DR-1α, together with a standardized comprehensive ocular surface/tear evaluation, was performed on 348 consecutive eyes. Investigator visually detected or software detected non-invasive tear break-up and NIBUT were measured and compared on a subset of these examinations. The relationship between software-detected NIBUT and categorical dry eye severity based on irritation symptoms and corneal and conjunctival dye staining scores was determined. The sensitivity of visual (frame-by-frame) or software detected non-invasive tear break-up in eyes with tear instability (FBUT < 10) was similar (range 63–69%). NIBUT, measured visually or by software, had a correlation coefficient of 0.87. NIBUT was significantly lower in severity levels 2 and 3 compared to levels 0 + 1, and level 3 was significantly lower than level 2. In conclusion, there is a good correlation between investigator visually detected and software-detected tear break-up and tear break-up time in the KOWA DR-1α interferometric fringe images. Software-detected NIBUT is a clinically relevant measure of dry eye clinical severity.