A High-Fat Diet (HFD) leads to disruption of mitochondrial biogenesis and dynamics. Exercise training, especially High-Intensity Interval Training (HIIT) increases mitochondrial biogenesis and dynamics. The present study aimed to investigate the effect of a period of HIIT with and without HFD consumption on the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1-α), Mitofusins-2 (Mfn2), Optic atrophy-1 (Opa1), Dynamin-related protein-1 (Drp1) and mitochondrial Fission protein-1 (Fis1) genes as indicators of mitochondrial biogenesis and dynamics function in the soleus muscle of male Wistar rats. Twenty-four healthy male Wistar rats were randomly divided into four groups: (1) Control, (2) Control + HIIT, (3) HFD, and (4) HFD + HIIT. The HIIT training protocol lasted for 10 weeks with a frequency of 3 sessions per week. The Real-Time Quantitative Reverse Transcription PCR method was used to investigate the gene expression. One-way ANOVA and Fisher's post-hoc analyses were used to examine group differences. HFD consumption caused an increase in weight (P <
0.05), the expression of Drp1 and Fis1 genes (P <
0.001), and a decreased expression of Pgc1-α, Mfn2, and Opa1 genes (P <
0.001). HIIT training increased the expression of PGC1-α (P = 0.009), Mfn2 (P <
0.004), and Opa1 (P <
0.011) genes, while it decreased the expression of Drp1 (P = 0.003) and Fis1 genes (P = 0.027). These findings suggest that HIIT can counteract the negative effects of HFD on mitochondrial function by modulating gene expression related to mitochondrial biogenesis and dynamics.