Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and immune staging. The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep) tubesmade no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) responses. Even agitated blood showed a trend towards reduced ELISpot responses and increased human leukocyte Ag (HLA) multimer readouts when stored for 18 h compared to 3 h. These changes were reduced by diluting blood prior to storage. Washing PBMCs with media containing 10% human serum increased PBMC yields by 40.5%, without affecting ELISpot responses and multimer counts. However, washes with > 10% human serum decreased multimer counts, with no additional improvement in PBMC yields. These findings may be relevant for optimizing and harmonizing PBMC processing procedures for T-cell assays.