Obesity affects roughly 42% of the US population. High fat/high sugar diets (HFHS) often referred as “western diet” contributes to this prevalence. Diet‐induced obesity (DIO) results in impaired metabolic responses and associated disease states (i.e., Type 2 Diabetes). Metabolic impairments in DIO are a result of changes in muscle metabolism and muscle fiber phenotype. This is determined by both mRNA expression and protein isoform‐content of myosin heavy chain (MHC). Fast muscle fiber phenotype (i.e., type IIb in mice) is characterized by lower capacity for lipid utilization, implicated in the pathogenesis of Type 2 Diabetes. Regular exercise expresses mRNA in a fiber type specific manner, shifting MHC proportions under healthy circumstances. However, diet and exercise‐driven fiber type shifts in DIO are less understood. We aimed to determine the impact of exercise and diet on fiber‐type proportions in mice with DIO. We hypothesized exercise shifts mouse gastroc muscle phenotype induced by HFHS diet away from IIb fast fiber types. 49 C57BL/6 mice were split into 4 groups: 1) Lean Control (n = 9) fed standard chow and water for 24 wks, 2) HFHS (n = 10), fed HFHS diet (60% of calories from fat, high sugar/fructose: 42 g/L in drinking water) for 24 wks, 3) HFHS Control (n = 10) fed HFHS diet for 12 wks followed by standard chow and water for 12 wks (i.e., simulating traditional diet approach), and 4) HFHS + exercise group (n = 10) fed HFHS diet for 24 wks and performed aerobic exercise (30 min of treadmill running 5 d/wk) in the last 12 wks. Gastroc muscles were collected, homogenized, and analyzed for MHC mRNA expression of MYH 1 (type IIx), MYH 2 (type IIa), MYH 4 (type IIb), and protein isoforms via q‐PCR and SDS‐PAGE respectively. Intensity of bands corresponding to MHC isoforms were quantified using Image J. mRNA expression was calculated using comparative CT analysis (2‐∆∆CT). Paired sample t‐tests were conducted to test differences between MHC isoforms across groups. Proportions of MHC IIb isoform increased (91 ± 3%) in HFHS compared to Control (81 ± 6.2%, p>0.05), HFHS Control (77 ± 3%, p=0.004), and HFHS + exercise groups (79 ± 5%, p=0.057). Additionally, MHC IIa/x proportions in the HFHS (8 ± 3%) compared to Control (17 ± 5.9%, p>0.05), HFHS Control (20 ± 2%, p=0.004), and HFHS + exercise (18 ± 4%, p=0.054) groups were reduced. There were no differences in mRNA expression data. These data suggest performing aerobic exercise with HFHS diet or switching to a standard diet restores muscle fiber phenotype in mouse gastroc. Yet, this was not reflected in mRNA expression data. Changes in MHC isoforms are likely better detected at the protein level rather than mRNA expression. We conclude that exercise and dietary intervention may be a good strategy to shift MHC isoforms away from extreme fast fiber phenotype with lower capacity for lipid utilization. Future research should aim to determine single muscle fiber phenotype shifts with long‐term diet and exercise in humans with obesity to better understand regulation of muscle fiber phenotypes impact on human metabolism.