Background: Most errors in the laboratory occur due to processes related to the pre-analytical phase. The purpose of this study was to investigate the effects of multiple freeze-thaw cycles on serum iron, transferrin, and ferritin, as well as the impact of short-term storage on the stability of these analytes. Methods: Serum samples from ten volunteers were submitted to three consecutive freeze-thaw cycles at -20°C and -80°C. Serum aliquots were also kept at 4°C, -20°C, and -80°C for 28 days. Iron, ferritin, and transferrin were measured after 1, 7, 14, 21, or 28 days of storage. Results: Serum ferritin and transferrin showed variations from -2.6 to 2.6% and -1.7 to 2.4%, respectively, in their concentrations during the three freeze-thaw cycles of -20°C and -80°C. However, the variations were statistically significant only at -20°C. No significant changes were found for iron at both temperatures. The storage at temperatures of 4°C, -20°C, and -80°C for up to 4 weeks significantly affected the serum concentrations of iron, ferritin, and transferrin (p < 0.01 for all). Conclusions: Serum iron, ferritin, and transferrin were affected by the storage of samples at temperatures of 4°C, -20°C and -80°C for up to 4 weeks, and the freeze-thaw cycles at -20°C influenced the measurement of serum ferritin and transferrin. [ABSTRACT FROM AUTHOR]