Abstract Background Total carbon dioxide (tCO2) measurements are important in the assessment of acid-base disorders. The collection and storage conditions of specimens are important preanalytical factors that can influence the accuracy of tCO2 measurements. Proficiency testing (PT) materials are used to assess the accuracy and reliability of a laboratory’s test performance. Unsuccessful performance on PT challenges can be an indicator of procedural issues. However, the stability of tCO2 in PT material compared to physiologic specimens is unknown and no guidance is provided to laboratories on how to handle PT samples for tCO2 measurement. This study aims to compare tCO2 stability in PT material with that of plasma samples when exposed to ambient air at room temperature. Methods Using enzymatic endpoint measurement on Vitros 4600 Chemistry System, tCO2 was measured in PT material (n = 5) and patient samples (n = 6) at baseline and after exposure to air for 15, 30, 60, 90, and 120 minutes. 500μL of each sample was aliquoted into EZ-nest tube adapters and baseline measurements were immediately analyzed. Average percent difference from baseline was calculated and >-10% bias was considered acceptable. Results The average percent difference from baseline in PT samples exceeded -10% as early as 30 minutes; whereas -3% bias was observed in patient samples at 60 minutes. Results are summarized in the table below. Conclusions The assessment of tCO2 stability ensures the reliability of laboratory measurements. Our results indicate tCO2 in PT material is not stable when exposed to air beyond 15 minutes, however tCO2 in most patient samples remained stable beyond 120 minutes. Understanding the stability limits of tCO2 in PT material allows us to devise workflows that prevent sample degradation during PT events. By establishing best practices for maintaining tCO2 integrity in PT material, laboratories can enhance the accuracy of proficiency testing results.