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Understanding variations in the routes by which wild animals gain and lose water is challenging, and common methods require longitudinal sampling, which can be prohibitive. However, a new approach usesΔ′¹⁷O_BW(Δ′¹⁷O of animal body water), calculated from measurements ofδ′¹⁷O andδ′¹⁸O in a single sample, as a natural tracer of water flux.Δ′¹⁷O_BWis promising, but its relationship to organismal variables such as metabolic rate and water intake have not been validated. Here, we continuously measured oxygen influxes and effluxes of captive deer mice (Peromyscus maniculatus), and manipulated their water intake and metabolic rate. We used these oxygen flux data to predictΔ′¹⁷O_BWfor the mice and compared these model predictions withΔ′¹⁷O_BWmeasured in blood plasma samples. As expected,Δ′¹⁷O_BWpositively correlated with drinking water intake and negatively correlated with metabolic rate. All predictedΔ′¹⁷O_BW(based on measured oxygen fluxes) values differed from measuredΔ′¹⁷O_BWvalues by <30 per meg (mean absolute difference: 11 ± 9 per meg), suggesting high accuracy for this modelling approach because studies currently report a range of 300 per meg forΔ′¹⁷O_BWamong mammals, birds and fish.