To investigate the stability of glucagon-like peptide-1(GLP-1) and glucagon in plasma under short- and long-term storage conditions. Methods: Pooled human plasma (n=20), to which a dipeptidyl peptidase 4 (DPP-4) inhibitor and aprotinin were added, was spiked with synthetic GLP-1 (intact, 7-36NH2 as well as the primary metabolite, GLP-1 9-36 NH2) or glucagon. Peptide recoveries were measured in samples kept for 1 and 3 hours at room temperature or on ice, treated with various enzyme inhibitors, after up to 3 thawing/refreezing cycles, and after storage at -20 and -80C for up to 1 year. Results: Recoveries were unaffected by freezing cycles or if plasma was stored on ice for up to 3 hours, but were impaired when samples stood at RT for more than 1 hour. Recovery of intact GLP-1 increased by addition of a DPP-4 inhibitor (no ice), but was not further improved by neutral endopeptidase 24.11 inhibitor or an inhibitor cocktail. GLP-1, but not glucagon, was stable for at least 1 year. Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared to immediate analysis, regardless of storage time. Conclusion: Plasma handling procedures can significantly influence results of subsequent hormone analysis. Our data support addition of DPP-4 inhibitor for GLP-1 measurement as well as cooling on ice of both GLP-1 and glucagon. Freeze/thaw cycles did not significantly affect stability of GLP-1 or glucagon. Long term storage may affect glucagon levels regardless of storage temperature and results should be interpreted with caution.