Investigating respiratory fluxes is decisive for understanding the complex interplay between metabolic processes. Studies of cells and tissues with limited sample availability and low respiratory rates may benefit from small experimental volumes. We examined if the 0.5 mL chamber yields results consistent with the 2.0 mL chamber at identical sample concentrations used in high-resolution respirometry. The background O2 flux was 4-fold higher in 0.5 mL than 2.0 mL chambers at air saturation, but was reproducible allowing for accurate background correction. The optimal stirring speed was between 550 and 750 rpm in the 0.5 mL chamber. Respiratory fluxes in living cells, permeabilized cells, and isolated mitochondria were measured in parallel in the 0.5 mL and 2.0 mL O2k-chambers, using SUIT protocols with 14-20 titrations. O2 fluxes in the two chamber types were nearly identical. Therefore, the 0.5 mL O2k-chamber, requiring close to four times less sample than the 2.0 mL chamber, offers a significant advantage for studies with limited amounts of sample or low respiratory capacities.