Objectives Exercise-induced hypoalgesia (EIH) is characterized by an increase in pain threshold following acute exercise. EIH is reduced in some individuals with chronic musculoskeletal pain, although the mechanisms are unknown. It has been hypothesized that this may relate to whether exercises are performed in painful or non-painful body regions. The primary aim of this randomized experimental crossover study was to investigate whether the presence of pain per se in the exercising muscles reduced the local EIH response. The secondary aim was to investigate if EIH responses were also reduced in non-exercising remote muscles. Methods Pain-free women (n=34) participated in three separate sessions. In session 1, the maximal voluntary contraction (MVC) for a single legged isometric knee extension exercise was determined. In sessions 2 and 3, pressure pain thresholds (PPT) were assessed at the thigh and shoulder muscles before and after a 3-min exercise at 30 % of MVC. Exercises were performed with or without thigh muscle pain, which was induced by either a painful injection (hypertonic saline, 5.8 %) or a non-painful injection (isotonic saline, 0.9 %) into the thigh muscle. Muscle pain intensity was assessed with an 11-point numerical rating scale (NRS) at baseline, after injections, during and after exercises. Results PPTs increased at thigh and shoulder muscles after exercise with painful (14.0–24.9 %) and non-painful (14.3–19.5 %) injections and no significant between-injection EIH differences were observed (p>0.30). Muscle pain intensity was significantly higher following the painful injection compared to the non-painful injection (p<0.001). Conclusions Exercising painful muscles did not reduce the local or remote hypoalgesic responses, suggesting that the pain-relieving effects of isometric exercises are not reduced by exercising painful body regions. Ethical committee number S-20210184. Trial registration number NCT05299268.