Groundwater discharge to streams can be distributed variably in space due to the heterogeneous composition of the subsurface. Fiber-optic distributed temperature sensing (DTS) has been applied to detect and quantify spatially concentrated groundwater discharge to streams. However, a systematic uncertainty assessment for this approach with respect to changing boundary conditions is missing, and limits of detection are unclear. In this study, artificial point sources with controlled inflow rates to a natural first-order stream were used to quantitatively test the approach for inflow rates in the range of <1% to approximately 19% of upstream discharge and varying temperature differences between stream water and inflowing water. Even small inflow fractions down to approximately 2% of upstream discharge could be detected with the DTS. Inflow fractions calculated from DTS-based stream temperature observations and independently measured inflow temperatures were comparable to measured inflow fractions. Average uncertainty estimation based on the error propagation calculations ranged between 9% and 22% for experiments well above the detection limits of the DTS but ranged up to 147% for experiments close to the lower end of the detectable range.