Matrix stiffening and external mechanical stress have been linked to disease and cancer development in multiple tissues, including the liver, where cirrhosis (which increases stiffness markedly) is the major risk factor for hepatocellular carcinoma (HCC). Stiffness is correlated with cancer development and patient prognosis, and is used clinically in disease surveillance. However, patients with non-alcoholic fatty liver disease (NAFLD) and lipid-droplet-filled hepatocytes can develop cancer in non-cirrhotic, relatively soft tissue. Given the clear association between mechanical stress and cancer development in cirrhosis, we hypothesized that lipid droplet accumulation in NAFLD is a direct internal mechanical stress with similar effects to tissue stiffening. To test this hypothesis, we developed specialized image analysis programs to quantify the impact of lipid accumulation (Chapter 2). Then, by treating primary human hepatocytes with the mono-unsaturated fatty acid oleate and culturing them on different stiffness substrates, we showed that lipid droplets are intracellular mechanical stressors, associated with nuclear deformation, chromatin condensation, and impaired hepatocyte function (Chapter 3). We found that lipid accumulation disrupts normal cytoskeletal network organization, reducing cell traction forces and preventing proper stiffness sensing. Mathematical modelling of lipid droplets as inclusions that have only mechanical interactions with other cellular components generated results consistent with our experiments, further supporting the idea that these impacts are mechanical. Together, these data show that lipid droplets are intracellular sources of mechanical stress that should be considered when researching the pathogenesis of NAFLD (Chapter 4). These results are also broadly applicable to the field of mechanobiology, as we have shown that internal sources of mechanical stress can impact nuclear mechanosensing and gene expression.