This study examined the biomechanical and neuromuscular responses to a structured active seating protocol compared to traditional seating and standing workstation use. Twenty-four healthy participants (12 male, 12 female) performed computer tasks at each of the workstations for 30-minutes. The active seated workstation had participants actively plantarflex and dorsiflex the ankles in a “stepping” motion to the sound of a metronome operating at 40 beats per minute. The participants’ biomechanical and neuromuscular responses were recorded using electromyography (EMG);​ near Infrared Spectroscopy (NIRS);​ pressure mats;​ galvanic skin receptors;​ heart rate (HR) monitor;​ and subjective discomfort data (ratings of perceived discomfort- RPD) for each collection period. Statistical analysis was conducted using a repeated measures analysis of variance for within-task and between-workstation comparisons. A Tukey’s post hoc analysis was calculated for any significant findings. This study demonstrated that the active sitting protocol significantly increased blood oxygenation levels to the gastrocnemius.