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Background & Aims To study the change of the lipid layer thickness analysis in medical staff (MS) before and after work, and to explore the significance of measuring lipid layer thickness (LLT) respectively in four quadrants. Methods Ocular Surface Disease Index (OSDI) questionnaire and video display terminal using time for 55 MS were collected (the informed consent was obtained from all patients). Noninvasive tear break-up time, LLT, tear meniscus height (TMH), meibomian glands (MG) dropout, and blink pattern before and after work (worked for more than 4 h) of 110 eyes were measured by Gaush iDea Ocular surface analyzer. Lid margin abnormalities were evaluated by the slit-lamp microscopy. Results The average OSDI score of 55 MS was 25.68 ± 14.91. The average LLT of 110 eyes after work (65.12 ± 3.63 nm) was significantly reduced compared to before work (66.54 ± 4.16 nm), p < 0.05. The LLT in the superior quadrant was significantly thinner than that in the other three quadrants, p < 0.01. The average LLT was positively correlated with the LLT in the inferior (r = 0.822, p < 0.001), nasal (r = 0.261, p < 0.001), and temporal quadrant (r = 0.372, p < 0.001), while was negatively correlated with the MG dropout in lower lid (r = −0.209, p = 0.002). There was a significant correlation between the LLT in the inferior quadrant and the VDTt (r = −0.173, p = 0.01). The LLT of inferior quadrant were positively related to the TMH (r = 0.149, p = 0.027) and negatively related to MG dropout in lower lid (r = −0.162, p = 0.017). Conclusion The LLT significantly decreases after work in MS. The distribution of the lipid layer on the ocular surface is uneven. It is unreasonable for current detection instruments to measure the inferior quadrant LLT alone to represent average LLT.