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Photosynthesis and respiration determine the carbon and oxygen (O₂) balance of ecosystems. Current methods used to estimate ecosystem respiration (ER) do not include diel ER fluctuations, which limit testing predictions about short‐term drivers of ecosystem metabolism. Diel changes in δ¹⁸O_O22 can be used to estimate diel ER due to discrimination against δ¹⁸O_O22 during respiration. We monitored diel δ¹⁸O_O22, O₂, light, and water temperature in three Wyoming streams and measured respiration fractionation (α_R) against δ¹⁸O_O22 in dark benthic flow chambers in two streams. The ranges of measured and literature α_R values were used to estimate uncertainty in metabolism parameters associated with not measuring α_R directly. Daytime ER was 54–340% higher than nighttime ER using δ¹⁸O_O22, but diel ER parameter estimates were highly uncertain relative to traditional estimates of ecosystem …