Heart rate asymmetry (HRA) refers to how asymmetrically the acceleration and deceleration patterns in heartbeat fluctuations are distributed. There is limited evidence regarding HRA changes in athletes and their association with autonomic regulation. This study aimed to compare the short-term HRA of high-performance athletes and non-athletes during an autonomic function test by calculating relevant HRA measures. This exploratory study obtained beat-to-beat RR interval time series from 15 high-performance athletes and 12 non-athletes during a standardized autonomic function test. This test includes rest, postural change, controlled respiration, prolonged orthostatism, exercise, and recovery phases. The following HRA parameters were computed from the RR time series for both groups: asymmetric spread index (ASI), slope index (SI), Porta’s index (PI), Guzik’s index (GI), and Ehlers’ index (EI). We found significant differences (p < 0.01) in the mean value of several HRA parameters between athletes and non-athletes and across the autonomic function test phases, mainly in postural change and recovery phases. Our results indicate that high-performance athletes manifest a higher number and magnitude of cardiac decelerations than non-athletes after an orthostatic challenge, as indicated by GI and EI. In addition, lower HRA was found in athletes in the recovery phase than in non-athletes, as indicated by ASI.