It is generally assumed that sarcomere lengths (SLs) change in isometric fibres following activation and following stretch on the descending limb of the force–length relationship, because of an inherent instability. Although this assumption has never been tested directly, instability and SL non–uniformity have been associated with several mechanical properties, such as ‘creep’ and force enhancement. The aim of this study was to test directly the hypothesis that sarcomeres are unstable on the descending limb of the force–length relationship. We used single myofibrils, isolated from rabbit psoas, that were attached to glass needles that allowed for controlled stretching of myofibrils. Images of the sarcomere striation pattern were projected onto a linear photodiode array, which was scanned at 20 Hz to produce dark–light patterns corresponding to the A– and I–bands, respectively. Starting from a mean SL of 2.55±0.07 μm, stretches of 11.2± 1.6% of SL at a speed of 118.9± 5.9 nm s–1 were applied to the activated myofibrils (pCa2+ = 4.75). SLs along the myofibril were non–uniform before, during and after the stretch, but with few exceptions, they remained constant during the isometric period before stretch, and during the extended isometric period after stretch. Sarcomeres never lengthened to a point beyond thick and thin filament overlap. We conclude that sarcomeres are non–uniform but generally stable on the descending limb of the force–length relationship.