The principal objective of this paper is to develop a simple and rapid method of estimating levels of fatigue so that chronic fatigue can be prevented. Long-distance runners belonging to a successful corporate team (Group A; 25 males) and representative runners at the prefectural level (Group B; 14 males) participated in this study. We examined the effects of strenuous physical exercise on serum enzyme activity and the fatigue level felt by the runners (subjective fatigue).The following parameters were measured on two consecutive mornings during a training period: physical characteristics, serum-biochemistry using the dry-chemistry method, and subjective fatigue determined using the questionnaire regarding subjective symptoms authorized by the Japan Association of Industrial Health and the Profile of Mood State (POMS). Group A was divided into A-Senior (17 males; highest performance level) and A-Freshman (8 males) subgroups according to the length of employment within the corporation (one year or more and less than one year, respectively).The levels of serum aspartate aminotransferase (AST) and creatine kinase (CK) were significantly lower in the A-Senior group than the other groups and this group displayed the "iceberg" POMS profile at both examinations. Some significant correlations between the elements of POMS and serum enzyme activity levels were observed among all three groups during both examinations. The decline of serum CK levels tended to accompany a decrease in "Fatigue" according to POMS among 9 of 17 members of the A-Senior group. This tendency between the A-Senior and the A-Freshman groups statistically differed according to theX - square analysis.Our results suggest that the effects of physical stress on serum enzyme activity levels and subjective fatigue are affected by performance levels. Physical fatigue seemed to be reflected by serum CK levels. Monitoring subjective fatigue while measuring serum enzyme activity levels using the dry-chemistry method immediately provides clinical value to players and coaches on site, and should therefore help to prevent a shift from "overreaching" to "overtraining".