An adequate level of physical activity may maintain or promote work ability in aging workers. Project Active is a randomized trial comparing a Lifestyle physical activity program with a Structured exercise program in sedentary but healthy adults aged 35 to 60 years. Subjects in both groups received 6 months of intensive intervention followed by 18 months of active follow-up. The total number of subjects was 235, from which 80 subjects participated in the work ability assessment. Primary outcome measures were energy expenditure (kcal · kg−1 · day−1), cardiorespiratory fitness (peak oxygen uptake in ml · min−1 · kg−1), and the Work Ability Index. At 6 months, daily energy expenditure had increased significantly over baseline (mean ± SD, from 33.0 ± 0.9 to 34.4 ± 1.8 kcal · kg−1 · day−1) and was maintained over baseline at 24 months (34.0 ± 2.5 kcal · kg−1 · day−1). The significant increase in energy expenditure was observed particularly in moderate levels of activity. The average percentage of body fat was significantly higher at baseline compared with 6 months and 24 months. Peak oxygen uptake increased from baseline significantly during the first 6 months (from 29.6 ± 5.7 to 30.6 ± 6.3 ml · min−1 · kg−1) and decreased to the baseline level (29.1 ± 5.5 ml · min−1 · kg−1) at 24 months. At baseline, the average Work Ability Index was 44.2 ± 4.0, and it remained unchanged at 6 months (44.4 ± 3.9) and at 24 months (44.2 ± 3.1). In conclusion, a 2-year physical activity intervention increased daily energy expenditure, reduced body fat, and maintained peak oxygen uptake in healthy, middle-aged, sedentary subjects. The average Work Ability Index score at baseline was excellent and did not change during the 2-year physical activity interventions.