BACKGROUND: The conditions under which samples were collected, processed, and stored in biobanks may influence Alzheimer's disease (AD) biomarker levels. OBJECTIVES: This study aims to investigate whether a range of pre-analytical factors influence plasma levels of AD biomarkers. METHODS: Data were obtained from the ASPREE Healthy Ageing Biobank, a cohort of healthy community-dwelling older individuals aged 70+ years in Australia. Five biomarkers were measured using plasma from 11,868 individuals: phosphorylated-tau181 (p-tau181), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and amyloid-beta 42 and 40 (Aβ42/Aβ40). Linear regression examined the association between pre-analytical factors and biomarker levels. RESULTS: Participants were aged 70-96 years, and 54 % were female. The mean storage time for samples was 10.6 years (range: 7.7-13.5). Some significant associations were identified between pre-analytical factors and biomarkers, in particular for p-tau181, but the effect sizes were small. Weak negative associations were found between p-tau181 and the time from venepuncture to laboratory (transport) (β: -0.82, p = 0.03), laboratory processing to frozen storage (β:-1.56, p <
0.001), and total years of storage (β: -0.45, p = 0.007), while a positive association was found with intermediate storage at -20 °C/-30 °C compared to -80 °C (β: 2.24, p = 0.004). Longer fasting time was associated with higher levels of both NfL (β: 0.15, p <
0.001) and GFAP (β: 1.75, p <
0.001). CONCLUSION: Following standard operating procedures, AD biomarkers can be measured in plasma from biobanks stored for up to 13 years, with minimal impact from long-term storage or other pre-analytical factors.