Objective: The goal of this study was to evaluate the prevalence and change in perception of physical discomfort, including musculoskeletal discomfort, during the COVID-19 pandemic and to determine associations of demographic factors, telework activity, and home office characteristics with the highest prevalence of discomfort. Background: The COVID-19 pandemic forced a substantial increase in telework in many countries. The sudden change from a regular workplace to an improvised “home office” may have an impact on working conditions and physical symptoms of office workers. However, investigations in this area remain limited. Method: A cross-sectional study design was used to compare self-reported complaints of physical discomfort perceived from before with those during the pandemic. Associations between complaints and home office characteristics were investigated from 150 faculty and 51 administrative staff of an academic institution with an age range of 41.16 ± 10.20 (59% female). Results: A significant increase of physical discomfort was found during the pandemic period for head, eyes, hand, and upper back for both staff and faculty and neck, shoulders, elbows, and lower back for faculty only. Logistic regression analyses point to associations with the lack of a laptop stand, uncomfortable desk, poor lighting, and sitting time, among others. Conclusion: A high prevalence of physical discomfort was reported by teleworkers during the COVID-19 pandemic. Some home office characteristics were associated with these discomforts. Application: Some telework characteristics seem to be risk factors for physical discomfort. Consideration should be given to teaching best practices for workstation setup and/or conducting other preventive interventions in the work environment.