Previous studies have explored the effect of differing heat and relative humidity (RH) environments on the performance of multiple anaerobic high-intensity interval training (HIIT). Still, its impact on physiological responses and performance following aerobic HIIT has not been well studied. This study examined the effects of differing RH environments on physiological responses and performance in college football players following HIIT. Twelve college football completed HIIT under four different environmental conditions: (1) 25 °C/20% RH (Control group); (2) 35 °C/20% RH (H20 group); (3) 35 °C/40% RH (H40 group); (4) 35 °C/80% RH (H80 group). The heart rate (HR), mean arterial pressure (MAP), lactate, tympanic temperature (TT), skin temperature (TS), thermal sensation (TS), and rating of perceived exertion (RPE) were recorded continuously throughout the exercise. The heart rate variability (HRV): including root mean squared differences of the standard deviation (RMSSD)、standard deviation differences of the standard deviation (SDNN)、high frequency (HF), low frequency (LF), squat jump height (SJH), cycling time to exhaustion (TTE), and sweat rate (SR) were monitored pre-exercise and post-exercise. The HR, MAP, lactate, TT, Ts, TS, and RPE in the 4 groups showed a trend of rapid increase, then decreased gradually. There was no significant difference in HR, MAP, TT, or RPE between the 4 groups at the same time point (p > 0.05), in addition to this, when compared to the C group, the lactate, Ts, TS in the other 3 groups significant differences were observed at the corresponding time points (p < 0.05). The RMSSD, SDNN, HF, and LF levels in the 4 groups before exercise were not significantly different. The RMSSD and HF in the H40 and H80 groups were significantly decreased and other HRV indicators showed no significant difference after exercise. In sports performance measurement, the SJH and TTE were significantly decreased, but there was no significant difference in the 4 groups. The SR was no significant difference in the 4 groups after exercise. In conclusion, heat and humidity environments elicited generally greater physiological effects compared with the normal environment but did not affect sports performance in college football players.