Humans spend approximately 80–90% of their time indoors. In current practice, indoor temperatures in many buildings are controlled very tightly. However, allowing more variation in indoor temperature results in more energy-efficient buildings and could potentially improve human metabolic and cardiovascular health. Therefore, this study aimed to evaluate the effect of a drifting ambient temperature versus a fixed ambient temperature on thermal physiological parameters and subjective perception. A cross-over intervention design was conducted in 16 healthy men (age 26 ± 4 y; BMI 23.0 ± 1.7 kg/m2) between July 2018 and May 2019. All participants underwent two whole-day (8:30–17:00) experimental sessions, during which they were exposed to a drifting (17–25°C with a morning ramp of 2.58°C/h and afternoon ramp of -2.58°C/h) or constant ambient temperature (21°C) in randomized order. The experiments took place in respiratory chambers, which simulated a typical office environment and in which temperature conditions can be controlled accurately. Throughout the experimental sessions core and skin temperature, heart rate, blood pressure, energy expenditure as well as activity levels were measured. Subjective thermal perception, such as thermal comfort and sensation, was assessed by questionnaires every 30 min. Results reveal that energy expenditure was higher in the morning during the drifting session, which was accompanied by an increase in activity levels. Both drifting and fixed sessions were judged as comfortable although during the drift thermal comfort was lower in the morning and afternoon and higher during midday. The results indicate that a drifting ambient temperature can be applied in practice, and as such, can contribute to a healthier and more sustainable built environment. More research is needed to understand the role of a drifting temperature on the long term.