Objective: The objective of the study was to evaluate the differences between sitting on a stability ball and in an office chair in terms of trunk muscle activation and lumbar spine posture. Background: Stability balls have become increasingly popular as an alternative to office chairs to help reduce the prevalence of low back pain; however, little research has been conducted on their use as office chairs. Methods: The 14 participants (7 men, 7 women) were required to sit on both a stability ball and an office chair for 1 hour each while performing various computer workstation tasks throughout the sitting periods. The activation of eight muscles and lumbar spine posture were measured and analyzed. Results: Increased muscle activation in thoracic erector spinae (p = .0352), decreased pelvic tilt (p = .0114), and increased perceived discomfort (p < .0001) while sitting on the stability ball were observed. Conclusions: The small changes in biological responses when sitting on a stability ball as compared with an office chair, combined with the increased reported discomfort while on the ball, suggests its use for prolonged sitting may not be advantageous. Application: Prolonged sitting on a stability ball does not greatly alter the manner in which an individual sits, yet it appears to increase the level of discomfort. Therefore, it is important to fully explore a new chair design and consult scientific research before implementing its use.