Background Musculoskeletal complaints are associated with a large medical and societal burden. Although acupuncture is a frequently used therapy for musculoskeletal complaints, little is known about the effect on health-related quality of life (HRQoL). Objectives The aim of this study was to (i) compare the HRQoL of patients undergoing routine acupuncture treatment for musculoskeletal complaints with a Dutch population sample; (ii) investigate changes in HRQoL during the course of acupuncture treatment. Methods An observational study of 26 patients between 18 and 65 years of age in a single acupuncture practice was performed. HRQoL was measured on eight functional domains using a RAND-36 health survey at baseline and after six and 12 treatment sessions. Baseline RAND-36 scores were compared to data from a Dutch population sample (n=1063) using t test, and longitudinal data were analysed using repeated measurement analyses. Results At baseline, patients had significantly lower RAND-36 scores compared to the Dutch population sample for three domains: role-physical limitations (51.9 vs 79.4; p<0.001), bodily pain (49.3 vs 79.5; p<0.001) and social functioning (75.5 vs 86.9; p=0.005). During the course of treatment, RAND-36 scores increased significantly for five domains: physical functioning (79.3 vs 97.4; p<0.001), role-physical functioning (51.4 vs 94.1; p<0.001), bodily pain (47.3 vs 95.7, p<0.001), social functioning (74.5 vs 92.0, p<0.001) and vitality (69.1 vs 85.7; p<0.001). Conclusion The observed improvements in HRQoL suggest a subjective, clinically relevant, benefit of routine acupuncture therapy in treating musculoskeletal complaints.