BACKGROUND: In many cohort studies, biological specimens are being stored without specific plans for analyses. In the Norwegian Mother and Child Cohort Study biological specimens (DNA, plasma, and whole blood) are stored on 96-well plates and as a result may undergo multiple freeze-thaw cycles. METHODS: To explore the impact of multiple freeze-thaw cycles on chemical constituents, we conducted a quality control study using pooled EDTA-plasma. Over a two-year period, samples stored at -80 degrees C were subjected up to 100 freeze-thaw cycles. Specimens were analyzed in triplicate for sodium, cholesterol, triglycerides, vitamin E, aspartate aminotransferase (AST), and free fatty acids. We assessed the percent change of analyte concentration from the values for the first freeze-thaw cycle, because this is the baseline for all stored specimens. RESULTS: With the exception of free fatty acids, there was little change over the first 10 freeze-thaw cycles. A majority of analytes showed no significant changes until 30 freeze-thaw cycles. After 30 freeze-thaw cycles, the largest percent change was observed for free fatty acids (+32%), AST (+21%), and triglycerides (-19%). CONCLUSIONS: Human plasma can go through several freeze-thaw cycles before analysis without influencing sample integrity for the selected analytes.