Evaluating leaf day respiration rate (R_L), which is believed to differ from that in the dark (R_Dk), is essential for predicting global carbon cycles under climate change. Several studies have suggested that atmospheric CO₂ impacts R_L. However, the magnitude of such an impact and associated mechanisms remain uncertain. To explore the CO₂ effect on R_L, wheat (Triticum aestivum) and sunflower (Helianthus annuus) plants were grown under ambient (410 ppm) and elevated (820 ppm) CO₂ mole fraction ([CO₂]). R_L was estimated from combined gas exchange and chlorophyll fluorescence measurements using the Kok method, the Kok-Phi method, and a revised Kok method (Kok-C_c method). We found that elevated growth [CO₂] led to an 8.4% reduction in R_L and a 16.2% reduction in R_Dk in both species, in parallel to decreased leaf N and chlorophyll contents at elevated growth [CO₂]. We also looked at short-term CO₂ effects during gas exchange experiments. Increased R_L or R_L/R_Dk at elevated measurement [CO₂] were found using the Kok and Kok-Phi methods, but not with the Kok-C_c method. This discrepancy was attributed to the unaccounted changes in C_c in the former methods. We found that the Kok and Kok-Phi methods underestimate R_L and overestimate the inhibition of respiration under low irradiance conditions of the Kok curve, and the inhibition of R_L was only 6%, representing 26% of the apparent Kok effect. We found no significant long-term CO₂ effect on R_L/R_Dk, originating from a concurrent reduction in R_L and R_Dk at elevated growth [CO₂], and likely mediated by acclimation of nitrogen metabolism.