Abstract Objectives Pre-analytical factors are a major source of variability in laboratory results. Failure to identify these factors can lead to falsely increased or decreased results and to erroneous clinical decisions. We aimed to investigate several pre-analytical factors influencing ammonia measurement, and to quantify their effect on the apparent increase in ammonia concentration. Design and methods Blood samples were taken from 20 healthy volunteers and submitted to five different sets of conditions: placing sample on ice vs. room temperature immediately after phlebotomy; centrifugation at room temperature vs. 0°C; measurement at 60min vs. 30min after sampling; storing sample at room temperature vs. 4°C; and use of stopper (i.e. open vs. closed tube). Ammonia was measured in all samples. Additionally, alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), free hemoglobin, and complete blood count were determined. Results Samples placed on ice immediately after centrifugation, samples spun at 0°C, and samples stored at 4°C all had lower changes in ammonia concentration than samples with less favorable treatments (P=0.008; P=0.033, and P=0.001, respectively). The observed biases exceeded clinically relevant acceptance criteria. Most of the tested parameters were significantly associated with increased ammonia. Multiple linear regression analysis identified only three variables that contributed significantly to the prediction of the dependent variable (i.e. increased NH3): ALT, GGT, and storage. Conclusion Pre-analytical factors cause significant errors in ammonia measurement. An increase in ammonia concentration is most strongly associated with ALT and GGT activity and with storage temperature.