Spores are of high interest to the food and health sectors because of their extreme resistance to harsh conditions especially against heat. Earlier research has shown that spores prepared on solid agar plates have a higher heat resistance than those prepared in a liquid medium. It has also been shown that the more mature a spore is the higher is its heat resistance contributed most likely by the progressive cross-linking of coat proteins. The current study for the first time enlightens the effect of sporulation medium on spore proteins. The 14N spores prepared on solid agar medium and 15N metabolically labelled spores prepared in liquid medium differ in their coat protein composition as revealed by LC-FT-MS/MS analyses. The 14N:15N isotopic ratio of the 1:1 mixture of liquid and solid medium cultured spores exposes that most of the identified inner coat and crust proteins are significantly upregulated while most of the outer coat proteins are significantly downregulated for the spores prepared on solid medium relative to the spores prepared in the liquid medium. Medium specific differences in isotopic ratios between the tryptic peptides of expected cross-linked proteins suggest that also the coat protein cross-linking may be medium specific. The spores prepared on solid medium show a higher thermal resistance than the spores prepared in liquid medium. Since the core DPA content is found to be similar in both the spore populations, it appears that the difference in wet heat resistance is connected to the differences in the coat protein composition and assembly. Supporting the proteomic analyses, the electron microscopy analyses show a significantly thinner outer coat layer of the spores cultures on the solid medium.