Evidence suggests that many employees spend most of their day sitting at their desk while using a computer workstation. Such sedentary behavior is linked with obesity, heart disease, and reduced arousal and mood. The current study aimed to expand existing research on the benefits of an active workstation for reducing sedentary time at work and improving employee occupational health outcomes. We conducted a within-subjects experiment in which 25 university employees used a treadmill workstation for an hour on three workdays and worked at their desk as usual for three additional workdays (with the order of conditions counterbalanced). At the end of each workday, participants completed measures of vigor, inattention, mood, job satisfaction, self-perceived performance, and physical health symptoms. We also gathered step count through pedometers that participants wore throughout the day. Results indicated that participants reported significantly higher levels of physical, cognitive, and emotional vigor and positive affect, and lower levels of negative affect and inattention on days when they used the treadmill workstation than on days when they worked at their desk as usual. However, we found no significant differences in job satisfaction, physical symptoms, nor self-perceived performance. We found that participants walked an average of 4500 more steps (similar to 2 miles) on days when they used the treadmill workstation as compared to their desk as usual. These findings suggest that using a treadmill workstation may have beneficial effects on employee well-being and physical activity while not detracting from performance.