Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m-cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4°C, 25°C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.g., the parabens and chlorobutanol were least destabilizing). We used hydrogen exchange-mass spectrometry (HX-MS) to better understand the destabilizing effects of temperature and preservative on backbone flexibility of AH-adsorbed P[4]. Thimerosal addition immediately increased the backbone flexibility across much of the AH-adsorbed P[4] protein backbone (except the N-terminal P2 region and residues G17-Y 38 ), and further increase in P[4] backbone flexibility was observed after storage (4°C, 4 weeks). HX-MS analysis of AH-adsorbed P[4] stored for 4 weeks at 25°C revealed structural alterations in some regions of the epitope involved in P[4] specific mAb binding. These combined results are discussed in terms of a generalized workflow for multi-dose vaccine formulation development for recombinant protein antigens.