The purpose of this study was to investigate changes in gait patterns, motor dexterity, cognition, neuromuscular performance, neural activation of the prefrontal cortex, and perception of effort and exertion during a 60-min walking trial at a reduced body weight using a positive pressure treadmill (PPT). Eleven screened astronaut analog participants performed 60-min PPT sessions at either 100% (PPT100) or 20% body weight (PPT20) which were separated into six 10-min segments. During each segment, a 1-min gait analysis, Pegboard Task, Assembly Task, and Memory Recall Task were performed. Step frequency, rating of perceived physical and cognitive exertion, electromyographic amplitude and frequency from the vastus lateralis, metabolic markers, and functional near-infrared spectroscopy (fNIRS) of the prefrontal cortex were obtained for all segments during each condition. The primary findings of the study indicated that PPT20 was able to induce differences in step frequency relative to PPT100 and that PPT100 resulted in greater metabolic expenditures and electromyographic amplitude compared to PPT20. The PPT20, however, exhibited increased fNIRS prefrontal cortex activity during the Assembly Task and Memory Recall compared to the PPT100. In addition, the PPT20 resulted in an increase in Memory Recall errors compared to PPT100. In conclusion, the findings of the present study indicated that altered gait patterns associated with reduced weight does not impact basic motor dexterity tasks, such as gait and Pegboard Tasks, but does increase the difficulty of Assembly Tasks and Memory Recall in astronaut analogs. Thus, novel gait patterns can impact the cognitive loads that astronaut analogs can perform. Special considerations should be given to the impact that gravity-induced changes in gait patterns have on motor and cognitive tasks in future planetary exploration missions.