This study was designed to examine the biomechanical and physiological responses between cycling on the Axiom stationary ergometer (Axiom, Elite, Fontaniva, Italy) vs. field conditions for both uphill and level ground cycling. Nine cyclists performed cycling bouts in the laboratory on an Axiom stationary ergometer and on their personal road bikes in actual road cycling conditions in the field with three pedaling cadences during uphill and level cycling. Gross efficiency and cycling economy were lower (-10%) for the Axiom stationary ergometer compared with the field. The preferred pedaling cadence was higher for the Axiom stationary ergometer conditions compared with the field conditions only for uphill cycling. Our data suggests that simulated cycling using the Axiom stationary ergometer differs from actual cycling in the field. These results should be taken into account notably for improving the precision of the model of cycling performance, and when it is necessary to compare two cycling test conditions (field/laboratory, using different ergometers).