Recently conventional fluorescent lighting has been replaced with LED color lighting in office environment because of high luminous efficacy and energy saving effect. Therefore, numerous studies have been widely performed to establish the design criteria and the standard of the performance. After reviewing the previous studies, we found that the previous studies mostly considered the factors of emotional stimulation as qualitative and subjective. Since the diverse wavelengths of LED color lighting can affect the human body in psychological and physiological ways it is essential to conduct a quantitative and objective study to investigate their effects. In this study, the objective was to verify the physiological effect of LED lights on the body. For this sake, LED (light emitting diode) flat lighting system was developed to have the size, 600×1200mm (L×H) and totally 100 LEDs were arranged. (five LEDs have 45mm intervals in width and twenty LEDs have 15mm invervals in length.) Ten males in their twenties were involved without the medical history of eyes and heart disorders. The subjects were exposed to LED lighting during five minutes and the experiment was conducted eight times changing with red, orange, yellow, green, blue, indigo, purple and colorless. Electroencephalogram (EEG), heart rate variability (HRV) and body temperature were measured before and after the stimulation. In case of EEG analysis, Relative power α wave ratio decreased in the groups of colorless, red and orange color lights. Also, sympathetic nerve was more activated than parasympathetic nerve and the body temperature was increased in the groups of colorless, red, orange, yellow color lights. On the other hand, relative power α wave ratio increased and parasympathetic nerve was more activated than sympathetic nerve and the body temperature was decreased in the groups of green, blue, indigo and purple color lights. The results of the study imply that the LED color lighting system in the realistic experiment environment. Using these results, they are expected to propose the color of LED lights to various working environments like study room or office etc. Also, they will help to extend the range of various fields of studies like using colors of LED lights to fields of design or treatment etc.