Physical inactivity impairs health by increasing morbidity. In childhood, modifiable risk factors associated with cardiovascular pathologies and related to mitochondrial function and structure are initiated by physical inactivity. The objective of this study was to analyze the effect of high-intensity swimming interval training (HIIT-swim) on cardiac mitochondrial ultrastructure in young Sprague Dawley rats compared with a sedentary group. Five-week-old Sprague Dawley rats (n = 18) were divided into a control group (C) (n = 6), a sedentary group (S) (n = 6) and an HIIT-swim group (H-s) (n = 6), the last of which performed HIIT-swim for 4 weeks. A mitochondrial ultrastructural evaluation was performed using transmission electron microscopy. In the H-s rats, mitochondrial areas and perimeters were found to be statistically significantly different from those of the C and S rats. In addition, no predominant intramitochondrial multifragmentation was observed in the mitochondria of H-s rats, but multifragmentation was evident in the mitochondria of S rats.