Left ventricular diastolic dysfunction is characterised by slow and delayed ventricular relaxation, increased chamber stiffness and is associated with significant morbidity and mortality. Over the physiological sarcomere length range the passive stiffness of cardiac muscle is predominantly mediated by the giant muscle protein titin. This study aimed to characterise the macro-molecular structure and nano-mechanical properties of titin populations by direct measurement of their axial mass and tensile strength. Titin was extracted and purified from ferret left ventricular tissue, air dried on carbon support films and unstained specimens were visualised by scanning transmission electron microscopy (STEM). By using the tobacco mosaic virus (TMV) as a standard of mass per unit length (MUL), precise measurements of MUL were made at regular intervals along native titin molecules to generate an axial mass distribution (AMD). Titin nano-mechanical properties were quantified by molecular combing¹ of partially adsorbed molecules on a poly-l-lysine modified mica surface. The surface tension force induced by the progression of the receding meniscus during air drying applies a tensile force of ~160pN to the adsorbed molecules. Combed and uncombed molecules were visualised by atomic force microscopy (AFM). Titin appeared as relatively straight filaments of variable diameter when adsorbed to hydrophobic carbon films and visualised by STEM. The axial mass distribution of these filaments fitted a biomodal Gaussian which peaked at 6.5 kDa/nm and 15 kDa/nm (n=280). Uncombed titin molecules, visualised by atomic force microscopy (AFM), appeared highly coiled. In contrast, combed molecules were straightened and aligned. Uncombed titin monomers ranged in length from 0.6 to 1.4 μm (n=40). Following molecular combing, Gaussian analysis revealed that monomer length increased from 1.2 to 1.7μm (n=42). Mean axial height was reduced following combing (0.62±0.21 nm vs 0.58±0.22 nm n=4000 p<0.01). These observations are in agreement with previous theoretical predictions of the mass density of titin monomers and hence dimers². The novel combination of STEM, molecular combing and AFM is capable of quantifying changes in titin structure and mechanical function induced by ageing and/or disease.