BACKGROUND Paraoxonase (PON1) enzymatic activity assays are used to characterize sensitivity to organophosphates and oxidative stress. Length of sample storage, temperature and other factors may influence variability of PON1 measurements, especially in longitudinal studies. METHODS Effects of assay temperature, storage duration up to 7 y (-80 degrees C), freeze-thaw cycles, the type of specimen (serum or heparinized plasma) and assay variability were evaluated for 4 PON1 substrate-specific assays using samples from two pediatric cohorts and laboratory volunteers. RESULTS Intra- and inter-assay variation, as well as inter-laboratory variability for PON1 activities were <10%. The effect of storage duration up to 2 y was minimal. However, after 7 y, arylesterase, paraoxonase, and chlorpyrifos-oxonase activities decreased more noticeably. Similarly, while freeze-thaw cycles did not affect the PON1 activities in samples stored <2 y, this factor was more significant after 7 y for arylesterase. Assay temperature and specimen type also influenced PON1 measurements. CONCLUSIONS Sources of technical variability of PON1 activity assays, including storage duration, freeze-thaw, and temperature should be monitored and minimized through study design, quality control procedures and statistical methods, especially in longitudinal studies where specimens may be stored for years prior to analysis.