Background: Hypoxic conditioning can help hikers adapt to low-oxygen conditions and hypoxic-loaded training can improve the physical fitness of hikers. Purpose: Study the effect of hypoxia conditioning on physiological parameters of acute altitude hiking. Methods: 24 healthy people with no high-altitude hiking experience and regular exercise habits (<2 times/week) participated in the hiking on the north peak of Hehuan Mountain (elevation 3422 meters). Participants were randomly assigned to either the hypoxia conditioning group (HC group, n=12, age 21.70 ± 2.42 years, height 165.75 ± 7.60 cm, weight 60.83 ± 10.24 kg) or the control group (CON group, n=12, age 21.39 ± 2.42 years, height 167.08 ± 9.27 cm, weight 62.16 ± 11.91 kg). All participants completed three treadmill workouts per week for four weeks, each consisting of 30 minutes of uphill walking at 15% grade, 8 kg load, and 3.6 km/h speed. The HC group additionally inhaled 13.5% oxygen during training sessions. Collect physiological parameters (Heart rate, HR; Rating of perceived exertion, RPE; Lactate, La; Oxygen saturation, SpO2; Acute mountain sickness, AMS; Hematocrit, Hct, and creatine kinase, CK) at different altitude points during the hike, as well as CK for five consecutive days after the end. Nonparametric statistics was used to compare within-group changes in each variable across time points. Kruskal-Wallis tests were performed for post hoc comparisons. Between-group differences were analyzed using Mann-Whitney U tests, with significance at p< .05. Results: Hypoxic conditioning enhanced altitude threshold height to a greater extent than regular training (p< .05). No significant differences (p>.05) were observed between the HC and CON groups in terms of physiological parameters during the hike. These did change as expected with uphill and downhill hiking, and altitude for oxygen saturation. Conclusions: This study explored how pre-hiking hypoxic conditioning (HC) affects hikers with no prior high-altitude experience. Compared to regular training, HC significantly improved altitude tolerance for these hikers. Both groups exerted themselves similarly during the hike. However, HC offered protection against altitude sickness despite similar exertion levels. This finding suggests HC as a valuable tool for hikers preparing for high-altitude ascents.