Although the physiological strain index (PhSI) is universal and comprehensive, its restrictions are recognized in terms of invasive on-site measurements and the requirement of accurate instruments. The perceptual strain index (PeSI) has been proposed as a user-friendly and practical indicator for heat strain. However, the application of this index in assessing the heat strain of construction workers has yet to be examined and documented. This study aims to ascertain the reliability and applicability of PeSI in an experimental setting that simulates a stressful working environment (i.e., environment, work uniform, and work pace) experienced by construction workers. Ten males and two females performed intermittent exercise on a treadmill while wearing a summer work uniform at 34.5°C and 75% relative humidity in a climatic chamber. Physiological parameters (core temperature, heart rate) and perceptual variables (thermal sensation, perceived exertion) were collated synchronously at 3min intervals. The results of two-way repeated measures analysis of variance (clothing×time) revealed that the PeSI was useful in differentiating the heat strain levels between different work uniforms. Not only did the PeSI change in the same general manner with the PhSI, but it was also powerful in reflecting different levels of physiological strain. Thus, the PeSI offers considerable promise for heat strain assessment under simulated working conditions.