Purpose The aim of this study was to confirm the impact of heat acclimation on aerobic performance in hot conditions and elucidate the transfer of heat adaptations to cool and hypoxic environments. Methods Ten males (VO2peak: 4.50 ± 0.50 L/min) completed two three-week interventions consisting of heat acclimation (HA: 36°C and 59% RH) and temperate training (TEMP: 18°C and 60% RH) in a counter-balanced crossover design. Training weeks consisted of four work-matched controlled heart rate sessions interspersed with one intermittent sprint session, and two rest days. Before and after the interventions VO2peak and 20-min time trial performance were evaluated in COOL (18°C), HOT (35°C) and hypoxic (HYP: 18°C and FiO2: 15.4%) conditions. Results Following HA, VO2peak increased significantly in HOT (0.24 L/min [0.01, 0.47], P = 0.040) but not COOL (P = 0.431) or HYP (P = 0.411), whereas TEMP had no influence on VO2peak (P ≥ 0.424). Mean time trial power output increased significantly in HOT (20 W [11, 28], P < 0.001) and COOL (12 W [4, 21], P = 0.004), but not HYP (7 W [−1, 16], P = 0.075) after HA, whereas TEMP had no influence on mean power output (P ≥ 0.110). Rectal (−0.13°C [−0.23, −0.03], P = 0.009) and skin (−0.7°C [−1.2, −0.3], P < 0.001) temperature were lower during the time trial in HOT after HA, whereas mean heart rate did not differ (P = 0.339). Conclusions HA improved aerobic performance in HOT in conjunction with lower thermal strain and enhanced cardiovascular stability (similar heart rate for higher workload), whereas the mechanistic pathways improving performance in COOL and HYP remain unclear.