A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated byex vivoanalysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenicE. coli(ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.