Atraumatically collected nonstimulated (less than 1 microliter/min) and stimulated (greater than 50 microliters/min) tears from 30 clinically normal subjects were fractionated by size exclusion high-performance liquid chromatography (SE-HPLC). Enzyme-linked immunosorbent assay (ELISA) and kinetic assays were applied to relevant HPLC fractions to quantitatively identify 12 tear proteins. Secretory IgA levels were much higher in nonstimulated than in stimulated tears, and a similar disparity was seen also with IgA1 and IgA2 in the HPLC fraction containing secretory IgA. IgM levels were also higher in nonstimulated tears. Levels of the primary lacrimal gland proteins, lactoferrin, tear specific prealbumin, and lysozyme were similar in both types of tears. Significantly higher concentrations of the major serum proteins, IgG, transferrin, and serum albumin were measured in nonstimulated tears. Overall, 8 of the 12 proteins assayed were present at significantly higher concentrations in nonstimulated tears. These results show that tear flow rate strongly influences the protein profile obtained. Therefore, to allow valid comparisons of tear protein profiles within and between studies that use atraumatic collection procedures, an indication of flow rate during collection should be reported.