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Abstract Background Low back pain (LBP) is one of the most disabling diseases and a major health issue. Despite the evidence of a link between paraspinal and gluteal muscle dysfunction and LBP, it is unknown whether aquatic exercises can lead to improvements in paraspinal and gluteal muscle morphology and function, and whether improvements in overall muscle health are associated with improvements in patients’ outcomes. The unique properties of water allow a water-based exercise program to be tailored to the needs of those suffering from LBP. This study uses magnetic resonance imaging (MRI) to investigate the effect of an aquatic exercise program versus standard exercise on 1) paraspinal and gluteal muscle size, quality and strength and 2) pain, disability, and psychological factors (pain related fear, depression, anxiety, sleep quality) in chronic LBP. Methods This study will include 34 participants with chronic non-specific LBP and moderate to severe disability, aged between 18 and 65, who will be randomly assigned (1:1) to the aquatic exercise group or land-based standard care exercise group. Both groups will receive 20 supervised sessions, twice per week over 10 weeks. MRIs will be obtained along the lumbosacral spine (L1-L5) and pelvis at the start and end of the intervention to assess the effect of each exercise intervention on paraspinal and gluteal muscle size and quality. Pre- to post-intervention changes in all outcomes between each group will be assessed, and the association between the changes in back muscle quality and clinical outcomes will be examined. Between-subjects repeated measure analysis of variance will be used to examine the changes in paraspinal muscle morphology over the different time points. Linear mixed models will be used to assess whether baseline scores can modify the response to the exercise therapy treatment. Discussion This study will determine if water-based exercises targeting the lower back and gluteal muscles can lead to important changes in muscle quality and function, and their possible relation with patients’ pain and functional improvements. Our findings will have strong clinical implications and provide preliminary data to design a community program to better support individuals with chronic LBP. Trial registration NCT05823857, registered prospectively on April 27th, 2023.