The study investigated how different storage conditions affect the quality of VPHP biological indicators (BIs). The focus was on the parameters D-value and Total Kill, which provide essential insights into the performance and reliability of the BIs. The investigation included tests under recommended and non-recommended storage conditions to evaluate potential impacts on the resistance and functionality of the BIs. The results of the study demonstrate and confirm the limit values for the storage of BIs, as well as the effects and consequently the possible errors of their use for the verification of e.g. cycle development or cycle validation.