αB crystallin and Hsp27 are small chaperone proteins that bind to unfolded proteins in order to block potential aggregation sites. Both are abundant cytoplasmic proteins in skeletal muscle fibres. Hsp70 is also a muscle chaperone protein, thought to be involved in folding of nascent and/or unfolded proteins. We hypothesised that unaccustomed exercise would lead to unfolding of proteins, which would be bound by these chaperones. P70S6kinase phosphorylation at thr 421/ser 424 is increased in type II fibres following resistance exercise, which is thought to reflect increased muscle protein synthesis1. Given that unfolded proteins are potentially associated with sites of repair, we hypothesised that fibres with increased chaperone binding would also show enhanced p70S6K phosphorylation. Biopsy samples were obtained from vastus lateralis muscle of healthy volunteers (n=8, age 21-35), unaccustomed to physical activity, before and after 3, 30 and 180 min of recovery following 30 min of treadmill running at a workload that elicited a steady-state plasma lactate concentration of 4 mM. Immunohistochemistry was subsequently performed on muscle cryostat sections, using classic double fluorescence, as well as in situ proximity ligation to demonstrate co-localisation. αB crystallin bound tightly and stably to insoluble structures, likely to be myofibrils and/or the cytoskeleton. To compare the extent of αB crystallin binding over the course of recovery, an arbitrary threshold was set just above the staining background of stitched micrographs covering whole sections and the fluorescence of the supra-threshold pixels expressed per muscle fibre area. Figure 1 shows that peak αB crystallin binding occurred after 3 min of recovery, and then declined to a value close to baseline after 3 hours. Staining was partially diffuse but also punctate, with strongly staining dots. Hsp27 also bound to the αB crystallin dots, in addition to intermediate filaments. In these dots, in situ proximity ligation co-localised the two small chaperones to within 20-40 nm, thus they are likely part of the same complex. The larger, strongest dots stained positive for Hsp70, where it also co-localised with αB crystallin. Given their established function, we interpret the co-localisation of three different chaperone proteins to the same site to indicate the presence of unfolded proteins. In fibres with numerous αB crystallin dots we found enhanced signals for p70S6K phosphorylation, resulting in a significant correlation between p70S6K phosphorylation and αB crystallin binding (p<0.05, r =0.57, Spearman). We believe this possibly reflects increased muscle protein synthesis at sites of protein unfolding.