Introduction: Complex mechanisms underlie dry eye (DE) symptom provocation. In particular, corneal hypersensitivity may provoke symptoms in short tear break-up time (BUT) DE characterized by tear film instability. We hypothesized that improved tear film stability may alleviate corneal sensitivity in patients with short tear BUT DE. Therefore, we investigated the effect of topical diquafosol tetrasodium (DQS) on corneal sensitivity in unstable tear film DE. Methods: This prospective, randomized study included 27 subjects (age: 39.1 ± 8.4 years; range: 25–59 years) with short tear BUT DE, defined based on the presence of DE symptoms and tear film instability. Subjects were randomly divided into DQS (3% DQS, 12 subjects) and artificial tear (AT; preservative-free AT, 15 subjects) groups. Subjects applied the medication 6 times a day for 5 weeks. The perception of touch (S-touch) and pain (S-pain) sensitivity was measured using a Cochet—Bonnet esthesiometer. Tear evaluation, corneal sensitivity, and DE symptoms were compared before and after DQS or AT administration. The correlation between the improvement degrees of corneal sensitivity and DE symptoms following medication was analyzed. Results: DQS significantly improved tear BUT and tear meniscus height (TMH) scores (p < 0.05), while AT significantly improved tear BUT (p < 0.05) but not TMH score. Mean S-pain and DE symptom scores were lower after medication use in the DQS (S-pain and DE symptoms: p < 0.05) and AT groups (S-pain: p = 0.05; DE symptoms: p < 0.05). However, S-touch did not change significantly in either group. A positive correlation was observed between the improvement degrees of S-pain and DE symptoms in the overall subjects studied. Conclusion: Both DQS and AT alleviate corneal hypersensitivity and DE symptoms in eyes with short tear BUT DE. However, DQS seems to be more effective to adjust tear environment, leading to the normalization of corneal sensitivity and DE symptoms. Trial Registration: UMIN Clinical Trials Registry Identifier, UMIN000014536.
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