Background: Six-minute walk test (6MWT) is a measure of functional capacity and a predictor of morbidity and mortality in the elderly. Published reference equations for the six-minute walk distance (6MWD) were derived from healthy subjects with sufficient physical activity, and may not be valid for patients with limited physical activity. Objective: To measure 6MWD in healthy individuals with sufficient (Suff) and insufficient (Insuff) levels of physical activity in Thai, and to compare the measured 6MWD with those calculated using published reference equations. Methods: Healthy volunteers aged 45-65 years (77 men and 85 women) performed three standard 6MWT. Physical activity levels were determined using a one-week recall physical activity questionnaire. The subjects were classified as having sufficient or insufficient levels of physical activity. Results: 6MWD of the Suff group was greater than the Insuff group in both men (701+89 m vs. 652+55 m, p = 0.005) and women (619+49 m vs. 571+35 m, p <0.001). The predicted 6MWD from published reference equations underestimated the measured 6MWD of men in Suff group, while it overestimated the measured 6MWD of women in Insuff group. Conclusion: Level of physical activity affected 6MWD of the subjects. Published references must be carefully applied for predicting 6MWD of individuals with various levels of physical activity.