Background Handrim wheelchair propulsion is often assessed in the laboratory on treadmills (TM) or ergometers (WE), under the assumption that they relate to regular overground (OG) propulsion. However, little is known about the agreement of data obtained from TM, WE, and OG propulsion under standardized conditions. The current study aimed to standardize velocity and power output among these three modalities to consequently compare obtained physiological and biomechanical outcome parameters. Methods Seventeen able-bodied participants performed two submaximal practice sessions before taking part in a measurement session consisting of 3 × 4 min of submaximal wheelchair propulsion in each of the different modalities. Power output and speed for TM and WE propulsion were matched with OG propulsion, making them (mechanically) as equal as possible. Physiological data and propulsion kinetics were recorded with a spirometer and a 3D measurement wheel, respectively. Results Agreement among conditions was moderate to good for most outcome variables. However, heart rate was significantly higher in OG propulsion than in the TM condition. Push time and contact angle were smaller and fraction of effective force was higher on the WE when compared to OG/TM propulsion. Participants used a larger cycle time and more negative work per cycle in the OG condition. A continuous analysis using statistical parametric mapping showed a lower torque profile in the start of the push phase for TM propulsion versus OG/WE propulsion. Total force was higher during the start of the push phase for the OG conditions when compared to TM/WE propulsion. Conclusions Physiological and biomechanical outcomes in general are similar, but possible differences between modalities exist, even after controlling for power output using conventional techniques. Further efforts towards increasing the ecological validity of lab-based equipment is advised and the possible impact of these differences -if at all- in (clinical) practice should be evaluated.