Background Relying on freezer stored biospecimens is preferred in epidemiolocal studies exploring environmental pregnancy exposures and later offspring health. Storage duration may increase the pre-analytical variability, potentially adding measurement uncertainty. We investigated evaporation of maternal serum after long-term biobank storage using ions (sodium, Na⁺; chloride, Cl^-) recognized for stability and relatively narrow normal biological reference ranges in human serum. Methods A chemical analysis study of 275 biobanked second trimester maternal serum from a large Danish pregnancy screening registry. Serum samples were collected between 1985–1995 and stored at -20°C. Ion concentrations were quantified with indirect potentiometry using a Roche Cobas 6000 analyzer and compared according to storage time and normal biological ranges in second trimester. Ion concentrations were also compared with normal biological variation assessed by baseline Na⁺ and Cl^- serum concentrations from a separate cohort of 24,199 non-pregnant women measured before freezing with the same instrument. Results The overall mean ion concentrations in biobanked serum were 147.5 mmol/L for Na⁺ and 109.7 for Cl^-. No marked linear storage effects were observed according to storage time. Ion concentrations were consistently high across sampling years, especially for specific sampling years, and a relatively large proportion were outside respective normal ranges in second trimester: 38.9% for Na⁺ and 43.6% for Cl^-. Some variation in concentrations was also evident in baseline serum used as quality controls. Conclusions Elevated ion concentrations suggest evaporation, but independent of storage duration in the present study (27–37 years). Any evaporation may have occurred prior to freezer storage or during the first 27 years. Other pre-analytical factors such as low serum volume have likely influenced the concentrations, particularly given the high within year variability. Overall, we consider the biobanked serum samples internally comparable to enable their use in epidemiological studies.