Multiple short daily bouts of HIIT are more effective than single daily sessions in improving cardiometabolic and cellular adaptations in rats. We hypothesize that a short period of detraining is sufficient to abolish the superior adaptive responses to multiple versus single daily sessions of HIIT in rats. Male rats were divided into untrained, 1xHIIT, and 3xHIIT groups. Over eight weeks, the 1xHIIT group performed 115 min single daily sessions of HIIT, while the 3xHIIT group performed three 5 min sessions with 4 h intervals. After training, both groups remained sedentary for four weeks (detraining). Resting oxygen consumption (VO2), body composition, glucose/insulin tolerance, and blood pressure were recorded. After euthanasia, cardiac function/histology and gastrocnemius mitochondrial density were analyzed. After training, both 1xHIIT and 3xHIIT protocols induced similar improvements in VO2, maximal oxygen uptake (VO2max), cardiac function/hypertrophy, and gastrocnemius mitochondrial density. These effects were maintained even after detraining. Only the 3xHIIT protocol improved insulin sensitivity. After detraining, this effect was abolished. After training, both 1xHIIT and 3xHIIT protocols reduced adiposity. After detraining, the adiposity increased in both groups, with a more pronounced increase in the 3xHIIT rats. A four-week detraining period abolishes the superior adaptive responses to multiple versus single daily HIIT sessions in rats.