Plant fibres are increasingly used as reinforcements, especially in thermoplastic composites. Understanding the impact of temperature on the properties of these fibres is an important issue for the manufacturing of high-performance materials with minimal defects. In this work, the structural evolution and mechanical behaviour of flax fibre cell walls were dynamically monitored by temperature-controlled X-ray diffraction and nanoindentation from 25 to 230 °C; detailed biochemical analysis was also conducted on fibre samples after each heating step. With increasing temperature up to 230 °C, a decrease in the local mechanical performance of the flax cell walls, of about −72 % for the indentation modulus and −35 % for the hardness, was measured. This was associated with a decrease in the packing of the cellulose crystal lattice (increase in d-spacing d200), as well as significant mass losses measured by thermogravimetric analysis and changes in the biochemical composition, i.e. non-cellulosic polysaccharides attributed to the middle lamellae but also to the cell walls. This work, which proposes for the first time an in-situ investigation of the dynamic temperature evolution of the flax cell wall properties, highlights the reversible behaviour of their crystalline structure (i.e. cellulose) and local mechanical properties after cooling to room temperature, even after exposure to high temperatures.