In this paper, we demonstrated that short-acting human insulin (HI) loses solubility when mixed with intermediate-acting HI. The recovery of soluble short-acting HI after having been mixed in varying ratios for different time periods with intermediate-acting HI was determined by radioimmunoassay. When regular HI (recombinant DNA): HI (rDNA) and NPH·HI (rDNA) were mixed in a 1: 1 ratio and then immediately centrifuged, there was no significant loss of regular HI (rDNA), although there was a significant loss (-43%) in the 1: 3 ratio. In contrast, there was a large and a significant loss (-71%, -91%) of Actrapid semisynthetic HI (SHI) in the 1: 1 and 1: 3 ratios when mixed with Monotard SHI. When regular HI (rDNA) and NPH·HI (rDNA) were mixed in 1: 1 and 1: 3 ratios after 20 min of incubation, there was a significant loss (-18%, -38%) of regular HI (rDNA). In contrast, there was a large and significant loss (-89%, -98%) of Actrapid SHI in the 1: 1 and 1: 3 ratios when mixed with Monotard SHI. These changes may affect the clinical bioavailability of short-acting HI in HI mixtures. Further studies are required to clarify the reasons for the mechanism of the loss of short-acting HI in HI mixtures.