Objective: The purpose of this study was to evaluate comfort levels of functional and non-functional chairs using subjective comfort rating, interface pressure measurement, muscle activity measurement, and skin temperature measurement. Background: Chairs are used for a prolonged period of time for sitting in many places such as the office, at university, at school, in industry, and so on. Almost all people use chairs in their everyday life. The functional properties of the chair are associated with comfort. Method: The subjective evaluation contains questions regarding chair comfort which can be rated with five point scale. The body-seat interface pressure was measured using a pressure mat system. The symmetry of sitting was measured using electromyography. The change in body part (thigh and buttock) temperature before and after sitting on a chair was measured with an infrared camera. Results: Participants rated significantly (p < 0.05) higher comfort scores for the functional chair in relation to the buttock and thigh region. Also, the participants felt a better cushion effect in the functional chair. When using the functional chair, lower interface pressure, better thermal comfort, and better symmetry of erector spinae muscle activity were observed. Conclusion: Overall, interface pressure measurement, muscle activity measurement, thermal imaging and subjective comfort score results showed that the functional chair was more comfortable than the non-functional chair. Application: The adopted methodologies could be used to measure the seating comfort of train seats.