Background Clinical trial samples may be stored frozen for prolonged periods before analysis, which can reduce the immunoreactivity of numerous analytes, particularly peptides. We sought to determine the effect of 6 years of frozen storage on serum N-terminal pro–B-type natriuretic peptide (NT-proBNP). Methods NT-proBNP was measured from serum samples taken from 99 different patients enrolled in the CanPREDDICT study after <1 year of storage at −70 °C using the Roche first-generation NT-proBNP assay on an e411 instrument. Separate aliquots of the same samples were analyzed after an additional 6 years of storage at −70 °C using the Roche second-generation assay on an e601 instrument. Results The median NT-proBNP immunoreactivity for the first measurement was 572 pg/mL (interquartile range [IQR] 205–1606, range 49–12820), while after an additional 6 years of storage at −70 °C, this value decreased to 526 pg/mL (IQR 181–1338, range 18–12880), resulting in a median percent difference of −7% (IQR −10.6% to −3.4%, P < 0.001). Conclusions We report findings consistent with trends seen in previous work but have investigated the effect of a much longer storage period. Larger percent decreases in NT-proBNP reaching statistical significance are seen, although the median difference is still <10%.