Plants have evolved several metabolic pathways as a response to environmental stressors such as low temperatures. In this perspective, it is paramount to highlight physiological mechanisms of plant responses to altitudinal gradients as a proxy to evaluate changing environments. Here, we aimed to determine the impact of elevation on the physiological attributes of two plant species along an altitudinal gradient. Our hypothesis was that the altitudinal gradient influences proline, protein, and sugar contents, as well as abscisic acid (ABA) and indole acetic acid (IAA) concentrations. We studied these physiological components in leaves collected from four different altitudinal ranges in Himalayan region of Pakistan from two native herbs, namely Bistorta affinis and Sibbaldia procumbens. Leaves were collected at the initial blooming phase from each altitudinal range, viz. 2850 m, 3250 m, 3750 m and 4250 m. We observed that most abiotic factors decrease with altitude which induces cold acclimation. A significant increase in the concentration of physiological components was observed as altitude increased, except for IAA, which decreased. Furthermore, we did not find variations in proline, ABA and IAA concentrations between species; only sugar and protein, with higher values for B. affinis. We conclude that altitudinal gradients significantly affect the physiological components of B. affinis and S. procumbens in Himalayan region. This result contributes to the understanding of how plants adapt to environmental pressures, acting as a proxy for the evaluation of impacts caused by climate changes.