Physical inactivity and low aerobic capacity are primary drivers of chronic disease pathophysiology and are independently associated with all-cause mortality. Conversely, increased physical activity and exercise are central to metabolic disease prevention and longevity. Although these relationships are well characterized in the literature, what remains incompletely understood are the mechanisms by which physical activity/exercise prevents disease. Given methodological constraints of clinical research, investigators must often rely on preclinical rodent models to investigate these potential underlying mechanisms. However, there are several key barriers to applying exercise metabolism findings from rodent models to human health. These barriers include housing temperature, nutrient metabolism, exercise modality, exercise testing, and sex differences. Increased awareness and understanding of these barriers will enhance the ability to impact human health through more appropriate experimental design and interpretation of data within the context of these factors.