Background: Storage of frozen plasma samples for hemostasis testing is a key step to obtain reliable results. Variables that can affect the quality of plasma during storage include the cryotube type and volume and the tube filling level that conditions the residual air volume. To date, there are only few data on which to base recommendations. Objectives: The aim of this study was to investigate the influence of the tube filling volume (20%, 40%, and 80%) of 2-mL microtubes on frozen plasma for a large panel of hemostasis assays. Methods: For this study, 85 subjects were included, and blood samples were collected from them by venipuncture. After double centrifugation, each sample was aliquoted in 3 2-mL microtubes with different volumes (0.4, 0.8, and 1.6 mL) and stored at - 80. C. At the end of the frozen storage period (3 months +/- 1 week), all aliquots from the sample were tested in the same analytical series for a large panel of hemostasis analyses. Results: Compared with completely filled microtubes (1.6/2 mL), storing frozen plasma in smaller volumes (0.4/2 mL) significantly decreased prothrombin time and activated partial thromboplastin time. Conversely, factor II, V, VII, and X levels were increased. Antithrombin, Russell's viper venom time, and anti-Xa activity in patients treated with heparin were also increased. Conclusion: To store plasma at -80. C for hemostasis analysis, samples should be frozen in small-volume microtubes (<2 mL) with screw caps that are filled to 80% of their volume.