AbstractLaboratory incubation of intact sediments is a popular and useful method for obtaining nutrient fluxes from lake sediments to the overlying water. However, one of the main problems facing this method is that the lake sediment core itself consumes oxygen from the overlying water due to the decomposition of organic matter. In this study, addition of headspace with controlled oxygen concentration and circulation of overlying water to the traditional intact sediment incubation method allows for incubation with a stable dissolved oxygen (DO) concentration in the overlying water. In 24 columns incubated for 179–269 h, the mean DO concentration in the overlying water ranged from 0.35 to 9.95 mgO2 L−1. In most cases, stability was high, with the standard deviation of the fluctuation in DO concentration less than 0.1 mgO2 L−1. The DO concentration of the overlying water could be adjusted by changing the partial pressure of oxygen in the headspace. This method supported a detailed description of the relationship between DO concentration and nutrient fluxes from sediments. The soluble reactive phosphorus fluxes and first‐order reaction constants of NO2− + NO3− fluxes increased rapidly when the DO concentration decreased below the thresholds of 1.5 and 3.0 mgO2 L−1, respectively, in Lake Biwa. The thresholds and precise relationships between DO concentration and nutrient fluxes provide useful information for predicting the effect of DO concentration decreases in the lake bottom on nutrient release from sediments. Our results indicate that this incubation method is a powerful tool for clarifying those relationships.