Numerous preanalytical variables (sample collection, pretreatment and storage conditions, miRNA extraction, etc.) can influence miRNA detection. Understanding the various properties of miRNA, especially its stability in biofluids, is important in various types of miRNA studies, both fundamental and applied. This study aimed to evaluate the influence of plasma storage conditions and certain RNA extraction parameters on stability of endogenous miRNAs in human blood plasma. We report stability kinetics of four endogenous miRNAs (-16, -19b, -23a, -451a) and cel-miR-39 as exogenous miRNA under short and long-term incubation at different temperatures as well as the effect of long-term storage on extracellular vesicles miRNAs stability. The most stable of the endogenous ones was miRNA-23a. When studying archival samples (1–2 and 9–10 years of storage) of blood plasma from healthy donors, it was shown, that the concentrations of all endogenous miRNAs are steadily decreasing. These findings further show that endogenous miRNA levels do not remain stable during prolonged storage at −20°C. Although packaging of miRNA in extracellular vesicles stabilizes miRNA to some extent, it nevertheless the level decreases over a period of time from 6 months to 6 years. We have also evaluated the effect of the reagents used in the extraction process on miRNA recovery. The addition of guanidine isothiocyanate containing denaturation buffer alone prevented degradation of the synthetic cel-miR-39 miRNA spiked-in to blood plasma. In the presence of denaturation buffer with or without 2-mercaptoethanol the yields were higher than with just 2-mercaptoethanol or in the absence of any agents. Addition of the commercial RNA-stabilizing agent RNAlater did not result in significant retention of miRNA in plasma, but significantly worsened the efficiency of miRNA isolation. Thus, the degradation rate of miRNAs can be affected by their structure and packaging. Addition of various stabilization solutions to biofluids can affect the efficiency of miRNA extraction.