Discrepancies in blood sample collection and processing could have a significant impact on levels of peptides in the blood, thus sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several pre-analytical processing conditions, including different storage times and temperatures of blood or plasma samples and different centrifugation forces, on the levels of peptides in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Both time and temperature were identified as major factors for peptide variation.