The study aimed to examine relationships between psychomotor fatigue threshold and the lowest heat stress limit (HSL) during incremental exercise, simulated in an environmental test chamber. Twenty-four soccer players performed a graded treadmill running exercise test. Directly before the test and during the break after each load, blood was collected to determine lactate concentration (LA) and serotonin concentration (SER). The heart rate (HR), pulmonary ventilation (Ve) and oxygen uptake (VO2) were recorded and the psychomotor test was performed. The levels of the tested parameters were determined at four measurement points: (1) at rest, (2) at the lactate threshold (TLA), (3) at the threshold of psychomotor fatigue (TPF), (4) at maximum intensity. Percentage differences between maximum intensity (100%) and the values of the tested parameters recorded at the TLA and TPF were also calculated. The tests were carried out in a climatic chamber at an ambient temperature of 28.5°C, relative air humidity of 58.7%. and wind speed of 2 m·s− 1. It was confirmed that the TPF, which reflects the highest efficiency of the central nervous system, occurs at a higher running speed than the TLA. For practical application, it was found that at the HSL, the TPF occurs at: 84% of maximum running speed, 52% of maximum LA concentration, 93% SERmax, 91% HRmax, 73% VEmax, 84% VO2max. The findings may facilitate the understanding of the physiological and psychomotor reactions at the borderline between some and great thermal discomfort (on the humidex scale). This will enable coaches and coaching staff to optimize training sessions in more challenging environments.