Background and Objective: Neutrophil extracellular trap (NET) formation has been proposed as a potential component of the inflammatory response associated with hemodialysis (HD). However, direct assessment of NET formation by neutrophils during HD has been limited. This study evaluated NET-associated signals in neutrophils during HD using flow cytometry and compared these findings with circulating NET-associated biomarkers. Methods: Ten clinically stable outpatients receiving regular HD with H₂-enriched dialysate (H₂-HD) participated in a prospective within-subject repeated-measures exploratory study. Patients underwent H₂-HD for 1 week (phase 1), standard HD (SHD) for 1 week (phase 2), and H₂-HD again for 1 week (phase 3). Blood samples were obtained immediately before and after the third HD session in each phase. NET-associated signals were assessed in isolated polymorphonuclear leukocyte preparations by flow cytometry using SYTOX Green and anti-myeloperoxidase (MPO) staining. Plasma cell-free DNA (cfDNA) and MPO-DNA complexes were also measured. Results: The proportion of SYTOX Green/MPO double-positive events in the predefined extracellular fraction increased significantly after HD during phase 2 (SHD), whereas no significant increase was observed during phases 1 or 3 (H₂-HD). Changes in the NET-associated signal did not differ significantly among the three phases. The change in the NET-associated signal was strongly correlated with the change in MPO-positive events (r=0.966, P<0.001), but not with changes in cfDNA or MPO-DNA complexes. Plasma cfDNA increased after HD before correction for hemoconcentration but not after correction. MPO-DNA levels decreased after HD after correction for hemoconcentration. Conclusions: Flow cytometry detected a neutrophil-associated NET signal that increased during SHD, providing evidence consistent with NET-associated extracellular DNA formation during HD. The discordant responses of flow cytometric and circulating NET-associated biomarkers suggest that these assays may reflect different aspects or temporal phases of the dialysis-associated neutrophil response. The effect of H₂-HD on NET formation remains uncertain.