The Tracer Additions for Spiraling Curve Characterization (TASCC) model has been rapidly adopted to interpret in‐stream nutrient spiraling metrics over a range of concentrations from breakthrough curves (BTCs) obtained during pulse solute injection experiments. TASCC analyses often identify hysteresis in the relationship between spiraling metrics and concentration as nutrient concentration in BTCs rises and falls. The mechanisms behind these hysteresis patterns have yet to be determined. We hypothesized that differences in the time a solute is exposed to bioactive environments (i.e. biophysical opportunity) between the rising and falling limbs of BTCs causes hysteresis in TASCCs. We tested this hypothesis using nitrate data from Elkhorn Creek (CO) combined with a process‐based particle tracking model representing travel times and transformations along each flow path in the water column and hyporheic zone, from which the bioactive zone comprised only a thin superficial layer. In‐stream nitrate uptake was controlled by hyporheic exchange and the cumulative time nitrate spend in the bioactive layer. This bioactive residence time generally increased from the rising to the falling limb of the BTC, systematically generating hysteresis in the TASCC curves. Hysteresis decreased when nutrient uptake primarily occurred in the water column compared to the hyporheic zone, and with increasing the distance between the injection and sampling points. Hysteresis increased with the depth of the hyporheic bioactive layer. Our results emphasize that good characterization of spatial heterogeneity of surface‐subsurface flow paths and bioactive hot spots within streams is essential to understanding the mechanisms of in‐stream nutrient uptake.