Individuals with poor metabolic flexibility are at elevated risk for cardiometabolic disease. Developing sensitive tests to detect poor metabolic flexibility before clinically significant metabolic disease has been established is vital to reduce disease risk. The aim of this study was to comprehensively assess fitness status and determine if high-intensity interval rowing exercise is sensitive enough to detect differences in metabolic flexibility between fitness statuses. The two groups significantly differed in terms of aerobic fitness, body composition, BMI, mitochondrial capacity, and amount of vigorous activity completed weekly. The groups were similar in all other assessed areas (dietary habits, quality of life, muscular strength, sedentary patterns, etc.). Consistent with the hypothesized outcome, groups were significantly different in substrate utilization during the high-intensity interval rowing exercise protocol. These findings suggest that the high-intensity interval rowing exercise protocol used in the present study was sufficient to detect differences in metabolic response between fitness statuses.