Subcutaneous adipose tissue lipolysis was studied in vivo by Fick's arteriovenous (a-v) principle using either calculated (microdialysis) or directly measured (catheterization) adipose tissue venous glycerol concentration. We compared results during steady-state (rest and prolonged continuous exercise), as well as during non-steady-state (onset of exercise and early exercise) experimental settings. Fourteen healthy women [age: 74 ± 1 (SE) yr] were studied at rest and during 60-min continuous bicycling at 60% of peak O₂ uptake. Calculated and measured subcutaneous abdominal adipose tissue venous glycerol concentrations increased substantially from rest to exercise but were similar both at rest and during later stages of exercise. In contrast, during the initial ~40 min of exercise, calculated glycerol concentration was significantly lower (~40%) than measured adipose tissue venous glycerol concentration. Despite several methodological limitations inherent to both techniques, the results strongly suggest that microdialysis and catheterization provide similar estimates of subcutaneous adipose tissue lipolysis in steady-state experimental settings like rest and continuous prolonged exercise. However, during shorter periods of exercise (<40 min), the results from the two techniques may differ quantitatively in the studied subjects. Caution should, therefore, be taken when lipolysis is evaluated, based on results obtained by the two techniques under non-steady-state conditions.