Plasma levels of circulating platelet extracellular vesicles (PEVs) are an emerging marker of platelet activation, thrombosis, inflammation, and endothelial dysfunction. Analysis of PEVs in cord blood of preterm newborns may reflect the underlying pathology and possibly serve as a new diagnostic and prognostic tool. However, collection, preparation and analysis of cord blood samples in clinical settings is a logistically complex process. We have studied the effect of delay in sample preparation and sample freezing on the PEV analysis by flow cytometry. PEVs in the cord blood plasma were identified after double labelling with monoclonal antibodies CD36+CD41 or CD41+CD62. Both, the delay and the freezing significantly affected the count and often also fluorescence of the detected PEVs. Additionally, our pilot study utilizing fresh cord blood samples of term and preterm newborns demonstrated significantly decreased CD36 and CD62 PEV fluorescence in preterm newborns. Our data highlight the importance of pre-analytical steps in the analysis of cord blood PEVs and suggest that not only the count, but also the level of PEV fluorescence may have possible diagnostic potential.