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The 21st mission of the National Aeronautics and Space Administration (NASA) Extreme Environment Mission Operations (NEEMO) was a highly integrated operational field test and evaluation of tools, techniques, technologies, and training for science driven exploration during extravehicular activity (EVA). The mission was conducted in July 2016 from the Aquarius habitat, an underwater laboratory, off the coast of Key Largo in the Florida Keys National Marine Sanctuary. An international crew of eight (comprised of NASA and ESA astronauts, engineers, medical personnel, and habitat technicians) lived and worked in and around Aquarius and its surrounding reef environment for 16 days. The integrated testing (both interior and exterior objectives) conducted from this unique facility continues to support current and future human space exploration endeavors. Expanding on the scientific and operational evaluations conducted during NEEMO 20, the 21st NEEMO mission further incorporated a diverse Science Team comprised of planetary geoscientists from the Astromaterials Research and Exploration Science (ARES/XI) Division from the Johnson Space Center, marine scientists from the Department of Biological Sciences at Florida International University (FIU) Integrative Marine Genomics and Symbiosis (IMaGeS) Lab, and conservationists from the Coral Restoration Foundation. The Science Team worked in close coordination with the long-standing EVA operations, planning, engineering, and research components of NEEMO in all aspects of mission planning, development, and execution.