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Purpose: To investigate the effects of humidity and temperature on dry eye disease (DED) Methods: A retrospective, clinic-based study was conducted on DED patients undergoing dry eye treatment. Patients were followed up at least twice, and symptoms and signs were evaluated using the Symptoms Assessment Questionnaire in Dry Eye (SANDE) score, tear secretion, tear film breakup time (TBUT), ocular staining score, and tear osmolarity. Mean humidity and temperature values for 1 week before ocular examinations were used as the environmental exposure level. The relationship between humidity and temperature, with DED clinical parameters was analyzed in single- and multi-environmental factor models. Results: The study included 33 patients with a mean age of 53.9 ± 12.2 years. The low humidity group showed significantly higher SANDE scores (p = 0.023) and tear osmolarity (p = 0.008), and the low temperature group had higher SANDE scores (p = 0.004), ocular staining scores (p = 0.036), and tear osmolarity (p < 0.001). In the linear mixed model, single factor analysis showed that an increase in humidity resulted in decreased SANDE scores (p = 0.043), and an increase in temperature led to a decrease in SANDE score (p = 0.007), ocular staining score (p = 0.007), and tear osmolarity (p = 0.012). In the multifactor analysis, changes in humidity had no significant effect on dry eye parameters, but an increase in temperature was significantly correlated with decreased SANDE score (p = 0.026), ocular staining score (p = 0.024), and tear osmolarity (p = 0.002). Conclusions: Lower temperature led to aggravated symptoms and signs of DED and the effect of temperature on DED was more pronounced than humidity. Tear osmolarity was the most sensitive clinical parameter to be affected by climate factors in DED patients.