Background: In this study the pre-analytical effects of sample storage on frequently used routine clinical chemistry assays were evaluated by comparing four different lithium heparin plasma separation tubes to a reference collection procedure. Methods: Blood was collected from 20 healthy volunteers using plasma separation tubes from four different manufacturers together with manually separated plasma as reference. In total, 15 clinical chemistry parameters were determined at 0 h, 24 h, and 72 h. Samples were stored at 48C. Statistical differences were evaluated using a generalized estimating equation regression model. Results: Significant differences could be demonstrated for almost every parameter when comparing the separation tubes to the reference collection system. The estimated maximum allowable storage time in the primary tube was considerably reduced using separation tubes, e.g., for glucose the maximum storage time was reduced from )72 h to 7-15 h, and for potassium from 60 h to 10-13 h, respectively. Conclusions: These data indicate that sample storage in the primary tube using plasma separation tubes is associated with clinically relevant changes for certain parameters. Therefore, storing samples for retesting should be avoided when using plasma separation tubes, in particular for parameters susceptible to interference by erythrocyte or platelet contamination.