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The mechanical unloading of spaceflight elicits a host of physiological adaptations including reductions in muscle mass, muscle strength, and muscle function and alterations in central interpretation of visual, vestibular, and proprioceptive information. Upon return to a terrestrial, gravitational environment, these result in reduced function and performance, the potential consequences of which will be exacerbated during exploration missions to austere and distant destinations such as the moon and Mars. Exercise is a potent countermeasure to unloading-induced physiological maladaptations and has been employed since the early days of spaceflight. In-flight exercise hardware has evolved from rudimentary and largely ineffective devices to the current suite onboard the International Space Station (ISS) comprised of a cycle ergometer, treadmill, and resistance exercise device; these contemporary devices have either fully protected or significantly attenuated neuromuscular degradation in spaceflight. However, unlike current microgravity operations on the ISS, future exploration missions will include surface operations in partial gravity environments, which will require greater physiological capacity and work output of their crews. For these flights, it is critical to identify physiological thresholds below which task performance will be impaired and to develop exercise countermeasures-both pre- and in-flight-to ensure that crewmembers are able to safely and effectively complete physically demanding mission objectives. © 2020 American Physiological Society. Compr Physiol 10:171-196, 2020., (Copyright © 2019 American Physiological Society. All rights reserved.)