Functional MRI of awake rats involves acclimatization to restraint to minimize motion. We designed a study to examine the effects of an acclimatization protocol (5 days of restraint, 60minutes per day) on the emission of 22-kHz ultrasonic vocalizations and performance on a forced swim test (FST). Our results show that USV calls are reduced significantly by day 3, 4 and 5 of acclimatization. Although rats show less climbing activity (and more immobility) in FST on day 5 compared to the 1(st) day of restraint acclimatization, the difference is gone once animals are given a 2 week hiatus. Overall, we show that animals adapt to the restraint over the five day period, however, restraint may introduce confounding behavioral outcomes that may hinder the interpretation of results derived from awake rat imaging. The present data warrant further testing of the effects of MRI restraint on behavior.