Recently, Live-Virtual-Constructive (L-V-C) integrate training system has proposed as a solution for the problems such as limitation of training areas, increase of mission complexity, rise in oil prices. In order to integrate each training system into the one effectively, we should solve the issue about stress of pilots by the environmental differences between Live and Virtual simulation which could be occurred when each system is connected together. Although it was already examined in previous study that the psychological effects on pilots was occurred by the environmental differences between actual and simulated flights, the study did not include what the causal factors affecting psychological effects are. The aim of this study is to examine which environmental factors that cause pilots’ psychological effects. This study analyzed the biochemical stress hormone, cortisol to measure the pilots’ psychological effects and cortisol was measured using Enzyme-linked immunoassay (EIA). A total of 40 pilots participated in the experiment to compare the differences in pilots’ cortisol response among live simulation, virtual simulation, and the virtual simulation applying three environmental factors (gravity force, noise, and equipment) respectively. As a result, there were significant differences in cortisol level when applied the gravity force and equipment factors to the virtual simulation, while there was no significant difference in the case of the noise factor. The results from this study can be used as a basis for the future research on how to make L-V system by providing minimum linkage errors and design the virtual simulator that can reduce the differences in the pilots’ psychological effects.