Cardiorespiratory fitness (CRF) has been reported to be inversely associated with visceral adipose tissue (VAT) accumulation, independent of body weight. However, the confounding effect of physical activity on the association between CRF and VAT remains inadequately addressed. On the basis of maximal oxygen uptake (VO2max), 143 sedentary, overweight women were dichotomized into high-fit (HF) and low-fit (LF) groups. Body composition and VAT were measured using dual-energy X-ray absorptiometry and computed tomography, respectively, and activity-related energy expenditure (AEE) was calculated using the doubly labeled water technique. No differences were observed between HF and LF for body mass index (HF 28.2±1.3; LF 28.3±1.31 kgm-2), total body weight (HF 77.5±6.8; LF 77.9±7.3 kg), total fat mass (HF 33.5±5.1; LF 33.9±4.4 kg) or AEE (HF 439.9±375.4; LF 517.9±298.7 kcal day-1). Significant differences in visceral adiposity (HF 68.5±30.4; LF 91.2±31.8cm2; P<0.001) and insulin sensitivity (HF 5.1±1.8; LF 3.1±2.4 SI ×10 -4 min-1 mIU-1 ml-1; P<0.01) were observed between the HF and LF groups, independent of age, race and AEE. This study affirms previous findings that CRF is an important determinant of the accumulation of VAT, and this relationship is independent of physical activity. © 2011 Macmillan Publishers Limited. All rights reserved.