This study was aimed to investigate the effects of acute and chronic High Intensity Interval Training (HIIT) on Irisin, BDNF (brain-derived neurotrophic factor), and Apelin levels. The study included twenty-one male soccer referees. Blood from the participants was collected at the beginning of study (1. first measurement: baseline value). HIIT was conducted and blood was immediately collected (2. second measurement: acute effect). Next, HIIT was carried out for 20 minutes of 4 days a week in bouts of running (75 meters in 20 seconds) and walking (25 meters in 20 seconds). Blood was collected at the end of 12 weeks (3. third measurement: chronic effect). HIIT was performed and blood was again collected (4. fourth measurement: acute effect after the chronic effect). There was a gradual increase in irisin, BDNF, and apelin levels (p < 0.001). The increase for irisin was 2% in the second measurement, 106% in the third, and 111% in the fourth compared to the first measurement. The increase for BDNF was 39% in the second measurement, 116% in the third, and 133% in the fourth. Apelin levels were increased by 11%, 19%and 28%, respectively. These results demonstrated that irisin and BDNF might increase only in response to chronic HIIT (4 times a week) while apelin levels might change with both acute and chronic HIIT in healthy trained referees.