Objectives: Non-medical hospital staff members are in frequent contact with patients and therefore are required to perform a wide variety of repetitive and high-frequency activities. The objective of this study was to assess the relationships between upper extremity activity and carpal tunnel syndrome (CTS) among non-medical hospital staff members. Material and Methods: Carpal tunnel syndrome in 144 non-medical hospital staff members was diagnosed using the Nordic Musculoskeletal Questionnaire (NMQ), a physician’s diagnosis, physical examination (Tinel’s signs and Phalen test) and a nerve conduction velocity (NCV) test. In addition, an ergonomic assessment was performed and a video camera was used to record the physical activities at work. Results: The prevalence rate of CTS was highest for the NMQ (51.9%), followed by physician’s diagnosis (49.5% for the right hand, 29.9% for the left hand), physical examination (54.7%), and nerve conduction test (motor nerve 27.5% and 25%, sensory nerve 21.7% and 15%, for right and left hands, respectively). Based on logistic regression models for the NMQ and physician’s diagnoses, there was a dose-dependently higher risk of CTS with the upper extremity index among participants, but this was non-significant based on the physical examination and nerve conduction tests. Conclusions: Nerve conduction velocity is the gold standard in diagnosis of CTS, but use of NMQ and physician’s diagnosis may overestimate the incidence of CTS in workers who have been engaging in repetitive stress activities for a relatively short time. Int J Occup Med Environ Health 2017;30(2):281–290