Objective: To investigate the influence of storage time and temperature on plasma insulin levels and to establish a correction formula. Methods: Venous blood samples were taken from 20 volunteers and processed as follows: whole blood samples, centrifuged samples, and separated plasma samples were stored at 4°C or 25°C. Insulin levels were determined by direct chemiluminescence at 0, 0.5, 1, 2, 4, and 8 hours. According to the correlation between the insulin concentration ratio and storage time, correction formulas for the insulin concentration were established. To verify the test, the venous blood samples of another 33 volunteers were processed in the same way. The insulin levels of the samples were corrected after 3, 6, 12, and 24 hours and compared with the value at 0 hours to verify the feasibility of the corrected formula. Results: With the prolongation of storage time, the insulin levels of the whole blood samples at 4°C or 25°C and of the centrifuged samples at 25°C decreased gradually (P <.001), and the insulin level correction formulas were Ccorrection = Cdetermination/0.991e−0.069 x, Ccorrection = Cdetermination/1.048e−0.126 x, and Ccorrection = Cdetermination/[−0.068ln(x) + 0.9242]. There was no significant difference between the corrected insulin results and the original results at any time within 12 hours (P >.05). Conclusions: The insulin levels of the whole blood samples at 4°C or 25°C and of the plasma samples at 25°C gradually decreased with storage time. The effect of storage time on the insulin level can be reduced with the correction formulas. © 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.