Manual restriction of head movement, or head-fixation, of awake rodents allows for sophisticated investigation of neural circuits in vivo, that would otherwise be impossible in completely freely moving animals. While it is known that head-fixation induces stress, the scale of this stress and habituation dynamics remain unclear. We used the Mobile HomeCage system (Neurotar Ltd, Finland) where animals have their heads fixed to an aluminum frame but are otherwise freely moving in an ultralight carbon container floating above an air-dispensing base. For 25 consecutive days, mice were head-fixed while standing on the air-lifted platform for 2 h per day and blood samples were taken periodically to measure variation in the stress-related hormone, corticosterone. We showed that the initial increase in corticosterone concentration is followed by a return to control level throughout the days of head-fixed training. We also found a locomotor correlate of this drop. We conducted a battery of stress-sensitive behavioral paradigms in freely-moving mice that revealed minor differences following chronic head-fixation. Finally, we analyzed motor-skill learning in the head-fixed setup with a floating container. We believe that our results may contribute to better interpretation of past literature and future in vivo experiments using head-fixed animals.