Satellite cells, the resident stem cells of skeletal muscle, are indispensible for muscle repair following injury and increasing evidence also points to an important role for other cell types, such as macrophages and fibroblasts, in orchestrating various stages of the repair process (Saclier et al., 2013, Murphy et al., 2011). However, the fibroblast content of human skeletal muscle at rest and during regeneration remains unknown. The purpose of this study was to determine temporal and spatial changes in the skeletal muscle content of satellite cells and fibroblasts during regeneration. The Regional Scientific Ethical Committees of Copenhagen approved this study (Ref: HD-2008-074), and all procedures conformed to the Declaration of Helsinki. 7 healthy males (age 21 ± 4 yrs; height 1.79 ± 0.03 m; weight 69 ± 8 kg (mean ± SD)) were subjected to 200 electrical stimulation (stim) induced eccentric contractions of the thigh muscles of one leg (Mackey et al., 2016). Muscle biopsies were collected from the vastus lateralis muscle of the stimulated leg 2, 7, and 30 days (d) post and from the unstimulated leg as a control. Fibroblast (TCF7L2+) and satellite cell (Pax7+) number were determined from immunohistochemically stained frozen sections. Data were analysed by repeated measures ANOVA and are reported as means ± SEM. In the control muscle, the number of satellite cells per fibre was 0.07 ± 0.004 and the number of fibroblasts 0.13 ± 0.02, resulting in a ratio of fibroblasts to satellite cells (f:sc) of 1.8 ± 0.2. Increases were observed on d7 and d30 for both cell types (p<0.05). Satellite cell number peaked on d7 at 0.23 ± 0.05 cells / fibre with a corresponding fibroblast number of 0.27 ± 0.04 cells /fibre. The proportion of damaged fibres on d7 was 25 ± 4%, which correlated strongly with the number of fibroblasts at this time point (Pearson’s r = 0.92, p = 0.003), where 57 ± 6 % of fibroblasts were found to be located immediately around these fibres. D30 fibroblast values were 0.45 ± 0.05 cells / fibre, a ~ 4-fold increase from control, where the f:sc ratio peaked at 2.7 ± 0.3, representing a 0.6-fold increase from control. The different temporal responses of satellite cells and fibroblasts observed during regeneration, with satellite cells peaking in number on d7 and fibroblasts during the later stages of repair (d30), is in line with persistent myogenic cell differentiation and extensive muscle extracellular matrix (ECM) remodelling at this stage (Mackey et al., 2016, Mackey et al., 2011) and warrants further investigation into the role of fibroblasts in regulating myogenic cell activity, as well as restoring the muscle ECM architecture, during the regeneration of human skeletal muscle.