Muscle size does not fully explain variations in muscle strength. Fat content has been implicated in muscle weakness, though this relationship remains unclear. The relationship between fat and strength may vary between scales (e.g., cellular, organ, and organism). The goal of this dissertation was to clarify the role of fat in the structure and function of muscle using in vitro and in vivo techniques across multiple scales in adults 21-45 years old. Study 1 tested the agreement of intramyocellular lipid (IMCL) content between oil red o (ORO) and magnetic resonance spectroscopy (MRS) techniques. These measures of IMCL were also compared to measures of quadriceps fat content (fat fraction, FF) by magnetic resonance imaging (MRI). Results showed that fat by ORO, MRS, nor MRI were related. This suggests that extracellular lipid contributes to IMCL by MRS, and that fat is not a primarily storage location within muscle cells. Study 2 quantified the relationships between FF, architecture, and strength in vivo using MRI, diffusion tensor imaging (DTI), and isokinetic dynamometry. There was a relationship between FF and fascicle length. However, FF was not related to measures of muscle strength. This suggests that fat may be related to shorter fascicles, but does not impair maximal strength. Muscle curvature and pennation angle were related to muscle strength, suggesting that they may be additional contributors to strength. Study 3 compared measures of single fiber function to whole muscle strength. The relationship between IMCL and single fiber function was also quantified. Measures of IMCL, FF, and BMI were not related, suggesting that IMCL does not contribute to fat t measured at the whole muscle or whole body levels. Measures of IMCL were also not related to single fiber function, indicating that greater levels of lipid accumulation may be necessary for fat-induced impairment of single fiber function. Collectively, the findings of this dissertation indicate that fat and mechanical function must be evaluated at the same anatomical scale for clear interpretations of their relationship. Additionally, this work suggests that fat may have a relationship with muscle structure, but not does have a direct effect on strength.