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In a cross-sectional study, surface electromyography measurements of the upper trapezius muscles were obtained during different functional tasks in patients with a chronic whiplash associated disorder Grade II and healthy control subjects.To investigate whether muscle dysfunction of the upper trapezius muscles, as assessed by surface electromyography, can be used to distinguish patients with whiplash associated disorder Grade II from healthy control subjects.In the whiplash associated disorder, there is need to improve the diagnostic tools. Whiplash associated disorder Grade II is characterized by the presence of “musculoskeletal signs.” Surface electromyography to assess these musculoskeletal signs objectively may be a useful tool.Normalized smoothed rectified electromyography levels of the upper trapezius muscles of patients with whiplash associated disorder Grade II (n = 18) and healthy control subjects (n = 19) were compared during three static postures, during a unilateral dynamic manual exercise, and during relaxation after the manual exercise. Coefficients of variation were computed to identify the measurement condition that discriminated best between the two groups.The most pronounced differences between patients with whiplash associated disorder Grade II and healthy control subjects were found particularly in situations in which the biomechanical load was low. Patients showed higher coactivation levels during physical exercise and a decreased ability to relax muscles after physical exercise.Patients with whiplash associated disorder Grade II can be distinguished from healthy control subjects according to the presence of cervical muscle dysfunction, as assessed by surface electromyography of the upper trapezius muscles. Particularly the decreased ability to relax the trapezius muscles seems to be a promising feature to identify patients with whiplash associated disorder Grade II. Assessment of the muscle (dys)function by surface electromyography offers a refinement of the whiplash associated disorder classification and provides an indication to a suitable therapeutic approach.