High altitude people required high endurance pack animals for load carrying and riding at prevalent mountainous terrains and rugged region. So far no studies have been taken to evaluate effect of loads on physiology of ponies in high altitude region. So, in this view we evaluated variation in physiological, hematological, biochemical, and cytokines indices of Zanskar ponies during load carrying at high altitude. Total twelve (12) numbers of Zanskar ponies, mare, age 4-6 years, were divided into three groups; group-A (without load), group-B (60 kg), and group-C (80 kg) of back pack loads. Track was very narrow and slippery with gravel, uneven with rocky surface and has a steep gradient of 4 km uphill at altitude 3291 to 3500 m. When we evaluate these parameters, it is understood that the heart rate, pulse rate and respiration rate was significantly (p<0.05) increased in 80 kg group among the three groups. The hematology parameters viz. hemoglobin, PCV, lymphocytes, monocytes %, ESR and eosinophil % significantly (p<0.05) changed in 80 kg group after load carrying among the three groups which was followed by control and 60 kg group. In biochemical parameters viz. LA, LDH, TP, HK, CORT, T3, CRT, AST, CK-MB, GPx, FRAP and IL-6 significantly (p<0.05) changed in 80 kg group after load carrying among the three groups which was followed by control and 60 kg group. The ALT, ALB, GLB, UR and UA significantly (p<0.05) changed in 80 kg group before and after load carrying among the three groups which was followed by control and 60 kg group. It has been concluded that, this result has revealed strong correlation of change in biomarkers level with performance in ponies during load carry. Hence, these parameters might be use for performance of endurance of Zanskar ponies in high mountain region.