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 δ18OO2 can be used to estimate diel ER due to discrimination against 18OO2 during respiration. We monitored diel δ18OO2, O2, light, and water temperature in three Wyoming streams and measured respiration fractionation (αR) against 18OO2 in dark benthic flow chambers in two streams. The ranges of measured and literature αR values were used to estimate uncertainty in metabolism parameters associated with not measuring αR directly. Daytime ER was 54-340% higher than nighttime ER using δ18OO2, but diel ER parameter estimates were highly uncertain relative to traditional estimates of ecosystem metabolism. Diel variations in water temperature only accounted for 4-55% of the range of diel ER calculated using diel δ18OO2. Measured benthic flow chamber αR varied within the range of literature values: from 0.9755 to 0.9954. Metabolism parameter estimates were very sensitive to choice of αR within the measured and published range of values. The mean and uncertainty of diel ER estimates increased with decreasing αR, with daily ER more than ten times higher given an αR of 0.975 vs. 0.999. Diel changes in ER can be modeled using δ18OO2 and O2, but diel ER estimates depend on the choice of αR, suggesting the need to better understand how αR may vary within spatial and temporal scales appropriate for δ18OO2 metabolism models.