Simulated hypoxic normobaric devices have been used in human beings in order to enhance endurance capacity. These devices are sealed chambers where the athletes are supposed to stay for at least 6–8 hr daily. The current research assesses the changes in time‐domain, spectral and non‐geometrical heart rate variability (HRV) parameters in 6 horses subjected to progressive duration periods inside of a hermetically sealed chamber. It was pursued, firstly to evaluate the intensity of the stress experienced by the animals and secondly to elucidate whether the horses might require an acclimation period before implementation of hypoxic conditions. HRV parameters were monitored for 6 days: day 0 (6‐hr duration; in paddocks; basal conditions), and days 1, 2, 3, 4 and 5 (1, 2, 3, 4 and 6 hr inside the chamber every day respectively). During day 1 and during the first hours of days 2 and 3, compared to day 0, horses presented increased HR and SDHR values and decreased RR interval duration. SD1 values decreased on some hours of days 2 and 3, but differences with day 0 were not found on day 1. Increased SDNN, RMSSD, SD1 and SD2 values were observed on days 4 and 5. These results showed an activation of the sympathetic activity together with an attenuation of the parasympathetic activity during the days 1 to 3. Increased parasympathetic activity was found only during the first hours of days 4 and 5. Spectral parameters experienced minor variations, with increased LFpeak and LF% during some hours of days 4 and 5. In conclusion, at least 3 days are needed to adapt the horse to a sealed environment before starting to subject the animals to hypoxic conditions. When the horses were acclimatized, however, a minor stress was detected with they spent more than 4 hr inside of the chamber.