Insulin formulations require careful storage temperatures. However, because insulin formulations are in the form of self-injectibles, patients must store them themselves. When patients store their insulin formulations, they are more likely to store them at temperatures other than those recommended by a medical facility. In fact, some patients leave their insulin formulations in their cars in the middle of summer, where temperatures can reach over 70°C. Therefore, we investigated the refolding limit temperature of insulin formulation under high temperature conditions based on secondary structure of insulin analogs. The results showed that human insulin, insulin glulisine, insulin lispro, insulin aspart and insulin degludec refolded at approximately 60°C. However, insulin detemir showed a change in the association state at temperatures lower than those of the other formulations, and insulin glargine showed cytotoxicity, even though the other parameters remained unchanged. These results indicate that insulin formulations may be used outside the recommended temperatures for short periods of time, such as when returning home from the pharmacy or during power outages. However, more attention should be paid to the storage temperature of insulin detemir and insulin glargine as compared to those of the other formulations. It is clinically difficult for patients to know the temperature and time of exposure; it is important to repeatedly instruct them to comply the recommended storage temperatures.