Objectives Retrospective studies frequently assume analytes long-term stability at ultra-low temperatures. However, these storage conditions, common among biobanks and research, may increase the preanalytical variability, adding a potential uncertainty to the measurements. This study is aimed to evaluate long-term storage stability of different analytes at <−70 °C and to assess its impact on the reference change value formula. Methods Twenty-one analytes commonly measured in clinical laboratories were quantified in 60 serum samples. Samples were immediately aliquoted and frozen at <−70 °C, and reanalyzed after 11 ± 3.9 years of storage. A change in concentration after storage was considered relevant if the percent deviation from the baseline measurement was significant and higher than the analytical performance specifications. Results Preanalytical variability (CV P ) due to storage, determined by the percentage deviation, showed a noticeable dispersion. Changes were relevant for alanine aminotransferase, creatinine, glucose, magnesium, potassium, sodium, total bilirubin and urate. No significant differences were found in aspartate aminotransferase, calcium, carcinoembryonic antigen, cholesterol, C-reactive protein, direct bilirubin, free thryroxine, gamma-glutamyltransferase, lactate dehydrogenase, prostate-specific antigen, triglycerides, thyrotropin, and urea. As nonnegligible, CV P must remain included in reference change value formula, which was modified to consider whether one or two samples were frozen. Conclusions After long-term storage at ultra-low temperatures, there was a significant variation in some analytes that should be considered. We propose that reference change value formula should include the CV P when analyzing samples stored in these conditions.