Aim Heat‐shock proteins (HSP) are important chaperones for stressed and damaged proteins. Low‐load blood‐flow‐restricted resistance exercise (BFRE) is generally believed not to induce significant muscle damage, but is hitherto unverified with intracellular markers. Consequently, the aim of this study was to investigate the HSP response after BFRE in human skeletal muscle. Methods Nine healthy volunteers performed five sets to failure of unilateral knee extension at 30% of 1RM with partial blood‐flow restriction. The contralateral leg performed the same work with free blood flow. Muscle biopsies were collected before exercise, 1, 24 and 48 h after exercise and analysed for HSP27, αB‐crystallin, HSP70, desmin, glycogen content and myosin heavy chain by immunohistochemistry, ELISA and western blotting. Results One hour after exercise, HSP27 and αB‐crystallin levels were reduced in the cytosolic and increased in the cytoskeletal fraction in the BFRE leg. HSP70 showed a delayed response and was increased over 48 h in the BFRE leg. Immunohistochemical analyses showed higher staining intensity of HSP70 in type 1 fibres in the BFRE leg at 24 and 48 h post‐exercise. PAS staining showed decreased glycogen levels after BFRE, and interestingly, glycogen was still depleted 48 h after exercise in the same fibres displaying high HSP70 staining (type 1 fibres). Conclusion Translocation of HSP27 and αB‐crystallin from cytosol to cytoskeletal structures indicates that cytoskeletal proteins are stressed during BFRE. However, overt signs of myofibrillar disruptions were not observed. Interestingly, the stress response was more pronounced in type 1 than in type 2 fibres and coincided with low glycogen levels.