So-called CO2 flux partitioning algorithms are widely used to partition the net ecosystem CO2 exchange into the two component fluxes, gross primary productivity and ecosystem respiration. Common CO2 flux partitioning algorithms conceptualise ecosystem respiration to originate from a single source, requiring the choice of a corresponding driving temperature. Using a conceptual dual-source respiration model, consisting of an above- and a below-ground respiration source each driven by a corresponding temperature, we demonstrate that the typical phase shift between air and soil temperature gives rise to a hysteresis relationship between ecosystem respiration and temperature. The hysteresis proceeds in a clockwise fashion if soil temperature is used to drive ecosystem respiration, while a counter-clockwise response is observed when ecosystem respiration is related to air temperature. As a consequence, nighttime ecosystem respiration is smaller than daytime ecosystem respiration when referenced to soil temperature, while the reverse is true for air temperature. We confirm these qualitative modelling results using measurements of day and night ecosystem respiration made with opaque chambers in a short-statured mountain grassland. Inferring daytime from nighttime ecosystem respiration or vice versa, as attempted by CO2 flux partitioning algorithms, using a single-source respiration model is thus an oversimplification resulting in biased estimates of ecosystem respiration. We discuss the likely magnitude of the bias, options for minimizing it and conclude by emphasizing that the systematic uncertainty of gross primary productivity and ecosystem respiration inferred through CO2 flux partitioning needs to be better quantified and reported.