We tried to measure fatigue levels by using several biological information, such as heart rates, flicker levels (F-values) or relative flicker value (ΔF-values) and ATMT (Advanced Trail Making Test) method. From the testing results, we found that there is a positive correlation between heart rates and F-values in the range of 'waking up' to 'awaking' stages, and the heart rates were independent of 'awaking' to 'sleep' stages. The result indicates that heart rates and F-values are related to fatigue levels. In addition, the maximum F-values and then slow decrease of F-value among the examinees correspond to 'perfect awakening' and fatigue levels in 'daily life'. Those results indicate that centrally acting fatigue is able to be measured with ΔF-value and physical fatigue levels were measured with ΔF-values when heart rates are approximately equal to before and after negotiating stairs. The F-values of the examinees decrease after consuming alcohols and then recover a few hours later, which were consistent with their blood alcohol levels. Therefore, the F-values are considered to be related with fatigue and blood alcohol level, because alcohol consumption causes indispensable symptoms of fatigue. As a result, we were able to measure various fatigue relating factors including centrally acting fatigue, physical fatigue and the fatigue of eyes, by using key biological information namely heart rates, F- or ΔF-values and ATMT method.