Background: It is increasingly common to collect and store specimens for future unspecified research. However, the effects of prolonged storage on the stability and quality of analytes in serum have not been well investigated. We aimed to determine whether the stability of liver enzymes extracted from frozen bio-samples stored at the baseline is affected by storage conditions. Methods: A total of four liver enzymes in the sera of 400 patients were examined following storage. After deter-mining the baseline measurements, the serum of each patient was aliquoted and stored at −70°C for three and six months, as well as one, two, and five years after collecting the original sample. The percent change from baseline measurements was calculated both statistically and clinically. Linear models were also used to correct the results of the samples based on the time they were frozen. Results: In almost all samples, liver enzymes were detectable until two years after the baseline, while in a signifi-cant proportion of samples, enzymes were not ultimately detectable five years after the baseline. Linear regression analysis on log-transformed levels of enzymes shows that the performance is acceptable until one year after the baseline. The performance of the prediction model declines substantially two and five years after the baseline, except for GGT. Conclusion: Long-term storage of serum samples significantly decreases the concentration of the liver enzymes from the baseline, except for GGT. It is not recommended to store samples for more than two years, as liver en-zymes are not detectable afterwards.