As Koreans habitually sit on the heated floor in their residential buildings, the male testis is directly exposed, and is therefore affected by the floor surface temperature. The purpose of this study was to investigate the changes in the scrotal temperature of the subjects in a sedentary posture over the heated floor. A rigid screening test was performed to select healthy subjects. Finally, six college students volunteered to participate in the experiments. Two experiments were performed in a controlled environmental chamber. Experiment I was designed for a low metabolism state, with the subjects reading a book in a sedentary posture for 50 min. The floor surface temperature (tf) was controlled by varying the temperature of water (tw) flowing into the floor coil from 15 to 50 °C, at 5 °C intervals. Experiment I revealed that the final scrotal surface temperature was 32.27, 32.62, 33.51, 33.34, 34.14, 34.28, 34.34 and 35.04 °C at the tw 15 °C (tf 17.0 °C), 20 °C (tf 20.8 °C), 25 °C (tf 24.1 °C), 30 °C (tf 27.8 °C), 35 °C (tf 31.7 °C), 40 °C (tf 35.9 °C), 45 °C (tf 38.6 °C) and 50 °C (tf 42.2 °C), respectively. At tf 17.0 and 20.8 °C, the scrotal temperature exhibited a declining pattern and a low temperature for spermatogenesis. At tf 24.1, 27.8 and 31.7 °C, however, the thermal regulatory system of the scrotum and testis was activated appropriately. On the contrary, scrotal temperature ascended at tf 35.9, 38.6 and 42.2 °C. Of the six subjects, two subjects demonstrated scrotal temperatures above 35 °C at tf 38.6 °C and four subjects exhibited scrotal temperatures above 35 °C at tf 42.2 °C. Experiment II was designed for a high metabolism state, with the subjects playing a card game in a sedentary posture for 180 min. The tf was controlled by varying the tw from 30 to 40 °C, at 5 °C intervals. Experiment II revealed that the final scrotal temperature was 33.43, 34.78 and 35.61 °C, and the difference between the initial and final scrotal temperatures was +0.34, +1.06 and +2.24 °C, at tw 30 °C (tf 27.8 °C), 35 °C (tf 31.7 °C) and tw 40 °C (tf 35.9 °C), respectively. The scrotal temperature was affected by the floor surface temperature and by the rate of metabolism of the subject in a sedentary posture. As derived from regression analysis, the recommended surface temperature of a heated floor is within 23–33 °C under the assumption that scrotal and consecutively testicular temperature above 35 °C impairs spermatogenesis.