Background: Nucleic acid test window periods for HIV, HCV, and HBV facilitate estimation of the residual risk of unexpected disease transmission and assist clinicians in determining the timeframe in which a recently acquired infection is at risk of nondetection. Objectives: Firstly, to provide revised estimates of the NAT window periods based on a currently used triplex NAT assay. Secondly, to examine their validity in organ donation and transplantation practice. Method: Estimates were based on the Procleix Ultrio Elite Assay (Grifols Diagnostic Solutions Inc. California, USA). The manufacturer's X50 and X95 limits of detection (LOD) were utilised. Viral doubling times of 0.85, 0.45, and 2.56 days and conversion factors for IU per ml to copies per mL of 0.6, 3.4, and 5 were assumed for HIV, HCV, and HBV respectively. Window periods were derived from the X50 and X95 LODs, based on a range of potential inoculum volumes. Results: Calculated X50 window periods were 5.1 (4.5–5.8), 2.7 (2.4–2.9), and 16.6 (14.2–19.1) days for HIV, HCV, and HBV respectively. Calculated X50 window periods, based on whole body plasma volume, were 11.8 (10.3–13.3), 6.2 (5.6–6.8) and 36.7 (31.3–42.1) days respectively. Conclusion: X50 NAT window periods were significantly shorter for HBV and HCV and sit at the lower range of previously published estimates for HIV. Current modeling assumptions may not account for all unexpected transmission events and may no longer be suitable for application to organ donation and transplantation. [ABSTRACT FROM AUTHOR]