Updated project metadata. Purpose: Determination of inter- and intra-day variations in tear flow rate, tear fluid protein concentration as well as protein composition regarding their impact for future biomarker studies. Methods: Tear fluid was collected non-invasively from 18 healthy subjects performing Schirmer tests at four different time points repetitive in a period of two days. The tear flow rate with Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. Results: Whether tear fluid was collected from OS or OD did not affect the tear flow rate (p ≈ 0.63) or protein concentration (p ≈ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume would only have an effect on total protein amount. When the examination days were compared, investigation of tear flow rate (p ≈ 0.001) and protein concentration (p ≈ 0.0003) indicated significant differences. Further mass spectrometric analysis of tear fluid revealed eleven differentially regulated proteins when comparing both examination days. Conclusion: Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration and composition in healthy subjects, suggesting that inter-day variation need to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.