Sit-stand desks continue to be a popular intervention for office work. While previous studies have reported changes in subjective measures, there is limited understanding of how sit-stand work differs from prolonged sitting or standing work, from a biomechanical standpoint. The objective of this study was to investigate the effects of prolonged sitting, prolonged standing, and a sit-stand paradigm on changes in trunk stiffness, low back discomfort, and trunk muscle activation. Twelve healthy participants performed 2 h of computer-based tasks in each protocol, on three different days. The sit-stand protocol was associated with a significant increase in trunk stiffness and a decrease in muscle activation of lumbar multifidus and longissimus thoracis pars thoracis, compared to both prolonged sitting and standing. Both sitting and standing were associated with increased low back discomfort. These findings may be worth exploring in more detail, for why alternating sit-stand patterns may help alleviate low back pain. Practitioner summary: We explored changes in objective and subjective measures related to low back discomfort following prolonged sitting, standing, and alternating sit-stand patterns. Alternating sit-stand pattern was associated with increased trunk stiffness and decreased back muscle activity. Hence, sit-stand desks may have benefits in terms of preventing/mitigating low back pain. Abbreviations: DOF: degree of freedom; EMG: electromyogram; ILL: iliocostalis lumborum pars lumborum; LTL: longissimus thoracis pars lumborum; LTT: longissimus thoracis pars thoracis; LBP: low back pain; LM: lumbar multifidus; MVEs: maximum isometric voluntary exertions; RANOVA: repeated-measure analysis of variance; RMS: root mean square