A series of experiments carried out in a controlled environment facility to induce steady-state chlorophyll a fluorescence variation demonstrate that natural fluorescence emission is observable on the derivative reflectance spectra as a double-peak feature in the 690-710 nm spectral region. This work describes that the unexplained double-peak feature previously seen on canopy derivative reflectance is due entirely to chlorophyll fluorescence (CF) effects, demonstrating the importance of derivative methods for fluorescence detection in vegetation. Measurements were made in a controlled environmental chamber where temperature and humidity were varied through the time course of the experiments in both short- and long-term trials using Acer negundo ssp. californium canopies. Continuous canopy reflectance measurements were made with a spectrometer on healthy and stressed vegetation, along with leaf-level steady-state fluorescence measurements with the PAM-2000 Fluorometer during both temperature-stress induction and recovery stages. In 9-h trials, temperatures were ramped from 10 to 35°C and relative humidity adjusted from 92% to 42% during stress induction, returning gradually to initial conditions during the recovery stage. Canopy reflectance difference calculations and derivative analysis of reflectance spectra demonstrate that a double-peak feature created between 688, 697 and 710 nm on the derivative reflectance is a function of natural steady-state fluorescence emission, which gradually diminished with induction of maximum stress. Derivative reflectance indices based on this double-peak feature are demonstrated to track natural steady-state fluorescence emission as quantified by two indices, the double-peak index (DPi) and the area of the double peak (Adp). Results obtained employing these double-peak indices from canopy derivative reflectance suggest a potential for natural steady-state fluorescence detection with hyperspectral data. Short- and long-term stress effects on the observed double-peak derivative indices due to pigment degradation and canopy structure changes were studied, showing that both indices are capable of tracking steady-state fluorescence changes from canopy remote sensing reflectance.