The master´s thesis deals with fatigue evaluation in electrooculography records (EOG). The theoretical part focuses on electrooculography itself, fatigue and methods used for fatigue detection from EOG. The practical part includes a plan of optimal methodology for fatigue evaluation using EOG. The EOG signals are recorded during the volunteers are watching prepared scenes. Those scenes are desribed here. There is also definition of signal processing methods with relevant block diagrams. Laboratory protocol describing EOG signals recording using Biopac data acquisition system is included. Ten volunteers were measured according to this protocol and the signal database was created. In brief questionnaire volunteers were supposed to evaluate fatigue and discomfort of the measurment. Recorded signals were processed and acquired parameters were statistically evaluated. Then the parameters were discussed in terms of fatigue detection ability. On the basis of that software application was created. This application detects fatigue in selected signal. The thesis includes detailed laboratory manual for students.