Alternate work positions are being considered as a way to address sedentary behaviour for office workers. This study evaluated the effect of two hours of just-sitting versus sitting while under-desk cycling on musculoskeletal discomfort and cognitive function (sustained attention and creative problem solving). To consider mechanisms, muscle fatigue, kinematics and mental state were also measured. Discomfort increased significantly across all body areas with knee and ankle discomfort greater (in a clinically meaningful manner) in under-desk cycling. Sustained attention reaction time was the only cognitive measure to show a difference between conditions (slower for under-desk cycling [β-34.82 CI (−62.12 to −7.53)]). There was no evidence of muscle fatigue, while kinematic differences between conditions were identified. Mental state deteriorated over time in both conditions. This study found no clear benefit of under-desk cycling compared to just-sitting for musculoskeletal health and cognitive function measures.