Three-helix bundles are a common repeat in natural proteins, and in a number of ways the helical bundles resemble soluble forms of membrane-spanning and other proteins. Spectrin is one such three-helix repeat protein and is a major component of the cytoskeleton that contributes to the stability and elasticity of the cell membrane. The properties of the repeats and their relative arrangement are key determinants of spectrin flexibility and can lend general insight into helical folding and bundling. Indeed, recent experimental studies on the thermal stability of spectrin repeats have revealed that eight of 36 such repeats melt as isolated repeats at temperatures below body temperature, and so these may be partially unfolded within the cell. Here we employ a combination of equilibrium and non-equilibrium molecular dynamics (MD) simulations with experimental techniques, such as circular dichroism, tryptophan fluorescence, cysteine labeling and atomic force microscopy, to help elucidate the key factors that determine the thermal and mechanical stability of spectrin repeats. The information provided from the natural variation of spectrin repeats should allow refinement of potentials and ensembles as well as provide insight for rational thermomechanical design of proteins.