To overcome the environmental changes occurring now and predicted for the future, it is essential that fruit breeders develop cultivars with better physiological performance. During the last few decades, high-throughput plant phenotyping and phenomics have been developed primarily in cereal breeding programs. In this study, plant reflectance, at the level of the leaf, was used to assess several physiological traits in five Vaccinium spp. cultivars growing under four controlled conditions (no-stress, water deficit, heat stress, and combined stress). Two modeling methodologies [Multiple Linear Regression (MLR) and Partial Least Squares (PLS)] with or without (W/O) prior wavelength selection (multicollinearity, genetic algorithms, or in combination) were considered. PLS generated better estimates than MLR, although prior wavelength selection improved MLR predictions. When data from the environments were combined, PLS W/O gave the best assessment for most of the traits, while in individual environments, the results varied according to the trait and methodology considered. The highest validation predictions were obtained for chlorophyll a/b (R2Val ≤ 0.87), maximum electron transport rate (R2Val ≤ 0.60), and the irradiance at which the electron transport rate is saturated (R2Val ≤ 0.59). The results of this study, the first to model modulated chlorophyll fluorescence by reflectance, confirming the potential for implementing this tool in blueberry breeding programs, at least for the estimation of a number of important physiological traits. Additionally, the differential effects of the environment on the spectral signature of each cultivar shows this tool could be directly used to assess their tolerance to specific environments.