In order to better understand the changes of body metabolism of athletes before and after exercise, this paper proposes a method of metabonomics feature analysis after exercise fatigue based on NMR. This paper makes a retrospective analysis on the metabonomic characteristics of urine of middle and long distance runners after 30 minutes of a heavy load training class based on NMR. The experiment analyzes the urine of athletes by combining NMR technology and metabonomics principle. The experimental results show that the Q2 value of this experiment is greater than that of all comparisons, and r2x and r2y are 0.436 and 0.85, respectively. Conclusion. This method can effectively analyze the changes of body metabolism after athletes’ fatigue, so as to better alleviate athletes’ fatigue after exercise.