Purpose: Closed eye neutrophils have demonstrated increased prevalence in dry eye disease, but the phenotype and extent of activation of these cells has yet to be described. Methods: 12 normal subjects and 12 subjects with dry eye disease were recruited and trained for self-collection of closed eye leukocytes, immediately upon awakening. Tear leukocytes were isolated and peripheral blood was collected, and stained with a panel of fluorescently-labeled antibodies to determine the activation phenotype of neutrophils. Extracellular matrix metalloproteinase 9 (MMP9) and neutrophil elastase (NE) was quantified by an enzyme-linked immunosorbent assay. Results: Total numbers of tear leukocytes recovered, at awakening, from normal and dry eye subjects were similar. Tear neutrophils from dry eye subjects had increased expression of membrane receptor CD66b, a marker associated with secondary granule degranulation. There was also a higher proportion of monocytes in the dry eye cohort, as compared to the normal cohort. Extracellular MMP9 was significantly higher in subjects with dry eye disease, and while NE was also elevated, it did not achieve statistical significance. Conclusions: Increased inflammation can be observed in the closed eye tears of subjects with dry eye disease, and neutrophils may be a potential source of pathogenic species in dry eye disease. Further research is required to determine the diagnostic potential of closed eye tears.