The accurate mechanical characterisation of fibres of micrometric length is a challenging task, especially in the case of organically-formed fibres that naturally exhibit considerable irregularities along the longitudinal fibre direction. The present paper proposes a novel experimental methodology for the evaluation of the local mechanical behaviour of organically-formed (aged and unaged) and regenerated cellulose fibres, which is based on in-situ micro-tensile testing combined with optical profilometry. In order to accurately determine the cross-sectional area profile of a cellulose fibre specimen, optical profilometry is performed both at the top and bottom surfaces of the fibre. The evolution of the local stress at specific fibre locations is next determined from the force value recorded during the tensile test and the local cross-sectional area. An accurate measurement of the corresponding local strain is obtained by using Global Digital Height Correlation (GDHC), thus resulting in multiple, local stress–strain curves per fibre, from which local tensile strengths, elastic moduli, and strains at fracture can be deduced. Since the variations in the geometrical and material properties within an individual fibre are comparable to those observed across fibres, the proposed methodology is able to attain statistically representative measurement data from just one, or a small number of fibre samples. This makes the experimental methodology very suitable for the mechanical analysis of fibres taken from valuable and historical objects, for which typically a limited number of samples is available. It is further demonstrated that the accuracy of the measurement data obtained by the present, local measuring technique may be significantly higher than for a common, global measuring technique, since possible errors induced by fibre slip at the grip surfaces are avoided.