The purpose of our study was to investigate the frequency of the third heart sound (S3) of athletes after exercise, and to determine whether the frequency and amplitude of S3 were related to cardiac function. The phonocardiogram exercise test (PCGET) was used in this study, and healthy volunteers consisting of 84 athletes (age 21.0±1.7 years; 62 males and 22 females) and 45 non-athletes (age 24.1±2.0 years; 33 males and 12 females) were enrolled. All subjects were healthy except one with a cardiac murmur without known cause. Immediately after exercise, S3 occurred in 21 athletes (25.0%) and 10 non-athletes (22.2%) during PCGET. There were very significant differences between pre-exercise and post-exercise in the frequency of S3 (P<0.01), and no significant difference between athletes and nonathletes (P>0.05). The prevalence of S3/S2≥1 was significantly (P<0.05) higher for the athlete group (47.1%) as compared to the non-athlete group (10%). Those results indicated that the emergence of S3 was an indicator of heart burden, and S3 after exercise in the athlete group was physiological. Our study showed that the amplitude of S3 had a very sensitive response to cardiac function reduction and S3/S2≥1 could eventually be used to assess cardiac fatigue states.