The purpose of this research is to clarify the level of fatigue in nursing students during classroom lecture, nursing seminar, and clinical practice from a physiological perspective. From 2010 through 2011 we conducted a survey of actual life situation and a questionnaire survey on a subjective symptom of fatigue on 59 second and third-year college nursing students. Also we measured vital signs, grasping power, and flicker fusion rate of these students as a physiological test. The survey of actual life situation showed that there was a difference on a period of time spent on hobbies, sleep, learning and computers between classroom lecture and nursing seminar. The analysis showed that grasping power which measures muscle function gradually declined on every form of learning from Monday through Friday. Its level was especially low during clinical practice. The average value of flicker fusion rate which measures mental and neurological function didn’t change depending other day of the week, but there was no statistically significant difference on the form of learning either. Attention concentration such as distraction and lack of patience and nursing seminar showed statistically significant difference on the association between a subjective symptom of fatigue and the form of learning. Also sleepiness and languor such as the languor of legs and clinical practice showed statistically significant difference on the association between a subjective symptom of fatigue and the form of learning. These findings show that it is necessary to find a better way to prevent and cope with fatigue symptoms for enhancing educational effects on nursing students.