Upward discharge to surface water bodies can be quantified using analytical models based on temperature–depth (T-z) profiles. The use of sediment T-z profiles is attractive as discharge estimates can be obtained using point-in-time data that are collected inexpensively and rapidly. Previous studies have identified that T-z methods can only be applied at times of the year when there is significant difference between the streambed–water interface and deeper sediment temperatures (e.g., winter and summer). However, surface water temperatures also vary diurnally, and the influence of these variations on discharge estimates from T-z methods is poorly understood. For this study, synthetic T-z profiles were generated numerically using measured streambed interface temperature data to assess the influence of diurnal temperature variations on discharge estimation and provide insight into the suitable application of T-z methods. Results show that the time of day of data collection can have a substantial influence on vertical flux estimates using T-z methods. For low groundwater discharge fluxes (e.g., 0.1 m d−1), daily transience in streambed temperatures led to relatively large errors in estimated flow magnitude and direction. For higher discharge fluxes (1.5 m d−1), the influence of transient streambed temperatures on discharge estimates was strongly reduced. Discharge estimates from point-in-time T-z profiles were most accurate when the uppermost point in the T-z profile was near the bed interface daily mean (two time periods daily). Where temperature time series data are available, daily averaged T-z profiles can produce accurate discharge estimates across a wide range of discharge rates. Seasonality in shallow groundwater temperature generally had a negligible influence on vertical flow estimates. These findings can be used to plan field campaigns and provide guidance on the optimal application of T-z methods to quantify vertical groundwater discharge to surface water bodies.