Glucose intolerance and diabetes mellitus are associated with physical inactivity, but it is unclear whether preventive interventions should aim at increasing overall energy expenditure or increasing participation in vigorous, fitness-enhancing activities. Studies aimed at separating and quantifying the effects of these two dimensions of physical activity should use well-validated measurement instruments and employ a study design in which the bivariate error structure of these instruments is determined. In the Isle of Ely Study (Cambridgeshire, United Kingdom), 775 individuals aged 45-70 years in 1994-1997 completed a glucose tolerance test and assessment of 4-day physical activity level (total energy expenditure/basal metabolic rate) by heart rate monitoring, a technique that has been validated against doubly labeled water and whole-body calorimetry. Cardiorespiratory fitness (maximum oxygen uptake (VO2max) per kg)) was measured in a submaximal test. To correct for measurement error, the authors had 190 individuals repeat both tests on three occasions at 4-month intervals. Two-hour glucose level was negatively correlated with physical activity level (men: r = -0.22, p < 0.001; women: r = -0.11, p < 0.05) and VO2max per kg (men: r = -0.18, p < 0.01; women: r = -0.19, p < 0.001) and was positively correlated with age and obesity. The model incorporating bivariate adjustment for measurement error showed that energy expenditure had a major effect on glucose tolerance, but there was less of an effect for cardiorespiratory fitness. These data provide support for public health strategies aimed at increasing overall energy expenditure.