Sustained mechanical loading of skeletal muscle may result in the development of a severe type of pressure ulcer, referred to as deep tissue injury. Recently it was shown that the diffusion of large molecules (10–150kDa) is impaired during deformation of tissue-engineered skeletal muscle, suggesting a role for impaired diffusion in the aetiology of deep tissue injury. However, the influence of deformation on diffusion of smaller molecules on its aetiology is less clear. This motivated the present study designed to investigate the influence of deformation of skeletal muscle on the diffusion of water, which can be measured with diffusion tensor magnetic resonance imaging (MRI). It could be predicted that this approach will provide valuable information on the diffusion of small molecules. Additionally the relationship between muscle temperature and diffusion was investigated. During deformation of the tibialis anterior a decrease of the apparent diffusion coefficient (ADC) was observed (7.2±3.9%). The use of a finite element model showed that no correlation existed between the maximum shear strain and the decrease of the ADC. The ADC in the uncompressed gastrocnemius muscle decreased with 5.9±3.7%. In an additional experiment a clear correlation was obtained between the decrease of the ADC and the relative temperature change of skeletal muscle tissue as measured by MRI. Taken together, it was concluded that (1) the decreased diffusion of water was not a direct effect of tissue deformation and (2) that it is likely that the observed decreased ADC during deformation was a result of a decreased muscle temperature. The present study therefore provides evidence that diffusion of small molecules, particularly oxygen and carbon dioxide, is not impaired during deformation of skeletal muscle tissue.