This pilot study questions the effects of a worksite physical activity program on health and fitness in tertiary employees. Ninety-five employees were randomly assigned to Control (CON); Novice (NOV); Experienced group (EXP). The NOV and EXP groups followed a 5-month worksite physical activity program (at least two sessions/week). Body composition, physical activity level and physical fitness, eating habits, health perception, sleep quality, pain, and quality of life were assessed. Fat mass decreased in NOV and EXP; the distance covered during the 6-minute walking test, push-ups, squat jump increased for NOV and EXP group. Physical activity level, health perception, quality of sleep, and eating habits were improved in NOV. This study underlines for the first time the beneficial effects of such worksite programs among tertiary employees on overall health and the feasibility of its design