Reducing sitting time by means of sit–stand workstations is an emerging trend, but further evidence is needed regarding their health benefits. This cross-sectional study compared work time muscle activity patterns and spinal shrinkage between office workers (aged 24–62, 58.3% female) who used either a sit–stand workstation (Sit–Stand group, n = 10) or a traditional sit workstation (Sit group, n = 14) for at least the past three months. During one typical workday, muscle inactivity and activity from quadriceps and hamstrings were monitored using electromyography shorts, and spinal shrinkage was measured using stadiometry before and after the workday. Compared with the Sit group, the Sit–Stand group had less muscle inactivity time (66.2 ± 17.1% vs. 80.9 ± 6.4%, p = 0.014) and more light muscle activity time (26.1 ± 12.3% vs. 14.9 ± 6.3%, p = 0.019) with no significant difference in spinal shrinkage (5.62 ± 2.75 mm vs. 6.11 ± 2.44 mm). This study provides evidence that working with sit–stand workstations can promote more light muscle activity time and less inactivity without negative effects on spinal shrinkage. Practitioner Summary: This cross-sectional study compared the effects of using a sit–stand workstation to a sit workstation on muscle activity patterns and spinal shrinkage in office workers. It provides evidence that working with a sit–stand workstation can promote more light muscle activity time and less inactivity without negative effects on spinal shrinkage.