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The primary aim of this study is to assess thermal comfort based on the predicted mean vote (PMV) and predicted percent dissatisfied (PPD) indices using subjective and experimental measurements in two air conditioned classrooms at a university, where the air-exchange rate is assured by natural ventilation. The indoor environmental conditions were satisfactory, and all situations fit within the comfort limits. The mean value of the PMV index ranges from 0.55 to −0.69 during both seasons, and the mean value of the PPD index ranges from 11.66 to 15.04%.The influence of the air conditioning system and ventilation provided by manually operated windows during the cooling season on thermal comfort parameters and CO2 concentration are also investigated by in situ measurements. In the absence of a cooling system and the ventilation rates, the air temperature exceeds the maximum comfort limit of 27 °C. Additionally, the PMV and PPD indices have the values of 0.87 and 21%, respectively and the CO2 concentration increases above the admissible limit, reaching a value of 2400 ppm. By manually opening the windows, the CO2 concentration decreases significantly to 1500 ppm. Thermal comfort is notably improved (PMV = −0.34, PPD = 7.4%) when the cooling system is running in the room. The secondary aim of this paper is to develop a prediction model of the academic performance during the cooling season. Application of this model indicates that the indoor environmental conditions can strongly affect student performance. Finally, a simulation model on the Transient System Simulation (TRNSYS) program of the PMV-PPD indices and heating/cooling energy demand for an amphitheatre with natural ventilation is proposed.