The 'Sit-Stand <i>e</i>-Guide' <i>e</i>-training program, designed to promote appropriate use of sit-stand workstations (SSWs), was evaluated for usability, acceptability and impact on various outcomes among SSW users. Participants from a large municipal organisation (25% male; mean age 45 [SD = 10.6] years) completed questionnaires pre-training (T0, <i>n</i> = 57), immediately post-training (T1, <i>n</i> = 50), and four weeks (T2, <i>n</i> = 46: primary endpoint) and twelve months later (T3, <i>n</i> = 30). High usability, acceptability and usefulness scores were reported at T1. Median [IQR] knowledge (4.4/5 [0.9]) and confidence (4.6/5 [1.0]) significantly increased at T1 compared to baseline (2.8 [1.2]; 3.3 [1.4]) and maintained at T2 and T3. At T2, mean [SD] sitting time (5.3 [1.2] h/workday) and low back discomfort (2.4 [2.3]) significantly decreased compared to baseline (6.1 [1.3] h; 3.4 [2.5] discomfort), SSW usage increased (1.4 [1.4]-2.8 [1.7] transitions), with no significant changes in work performance. Some behavioural changes were sustained at T3. The Sit-Stand <i>e</i>-Guide was acceptable and effective; evaluation across diverse workplaces and workers is now needed.