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IMPORTANCE There are limited reports on the relationship between mucin 5AC (MUC5AC) concentrations in tears, working hours, and the frequency of ocular symptoms in visual display terminal (VDT) users. This investigation evaluated these relationships among patients with dry eye disease (DED) and individuals serving as controls. OBJECTIVE To determine the relationship between MUC5AC concentration in the tears of VDT users based on the diagnosis of DED and frequency of ocular symptoms. DESIGN, SETTING, AND PARTICIPANTS An institutional, cross-sectional study was conducted. Participants included 96 young and middle-aged Japanese office workers. Both eyes of 96 volunteers (60 men and 36 women) were studied. Participants working in a company that used VDTs completed questionnaires about their working hours and the frequency of ocular symptoms. Dry eye disease was diagnosed as definite or probable, or it was not present. Tear fluid was collected from the inferior fornix after instillation of 50 μL of sterilized saline. The MUC5AC concentration was normalized to tear protein content and expressed as MUC5AC (nanograms) per tear protein (milligrams). The differences in MUC5AC concentration between DED groups, between VDT working hours (short, intermediate, and long), and between symptomatic and asymptomatic groups were evaluated with 95% CIs based on nonparametric Hodges-Lehmann determination. MAIN OUTCOMES AND MEASURES Ocular surface evaluation, prevalence of DED, and MUC5AC concentration. RESULTS The prevalence of definite and probable DED was 9% (n = 9) and 57% (n = 55), respectively. The mean MUC5AC concentration was lower in the tears of VDT users with definite DED than in those with no DED (P = .02; Hodges-Lehmann estimator, −2.17; 95% CI, −4.67 to −0.30). The mean MUC5AC concentration in tears was lower in the group that worked longer hours than in the group that worked shorter hours (P = .049; estimated difference, −1.65; 95% CI, −3.12 to 0.00). Furthermore, MUC5AC concentration was lower in participants with symptomatic eye strain than in asymptomatic individuals (P = .001; estimated difference, −1.71; 95% CI, −2.86 to −0.63). CONCLUSIONS AND RELEVANCE The data obtained in the present study suggest that office workers with prolonged VDT use, as well as those with an increased frequency of eye strain, have a low MUC5AC concentration in their tears. Furthermore, MUC5AC concentration in the tears of patients with DED may be lower than that in individuals without DED.