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Purpose: This study evaluated whether glycogen-associated water is a protected entity not subject to normal osmotic homeostasis. An investigation into practical and theoretical aspects of the functionality of this water as a determinant of osmolality, dehydration, and glycogen concentration was undertaken. Methods: In vitro experiments were conducted to determine the intrinsic osmolality of glycogen-potassium phosphate mixtures as would be found intra-cellularly at glycogen concentrations of 2% for muscle and 5 and 10% for liver. Protected water would not be available to ionic and osmotic considerations, whereas free water would obey normal osmotic constraints. In addition, the impact of 2 L of sweat loss in situations of muscle glycogen repletion and depletion was computed to establish whether water associated with glycogen is of practical benefit (e.g., to increase 'available total body water'). Results: The osmolality of glycogen-potassium phosphate mixtures is predictable at 2% glycogen concentration (predicted 267, measured 265.0 ± 4.7 mOsmol kg) indicating that glycogen-associated water is completely available to all ions and is likely part of the greater osmotic system of the body. At higher glycogen concentrations (5 and 10%), there was a small amount of glycogen water (~ 10-20%) that could be considered protected. However, the majority of the glycogen-associated water behaved to normal osmotic considerations. The theoretical exercise of selective dehydration (2 L) indicated a marginal advantage to components of total body water such as plasma volume (1.57% or 55 mL) when starting exercise glycogen replete. Conclusion: Glycogen-associated water does not appear to be a separate reservoir and is not able to uniquely replete water loss during dehydration. [ABSTRACT FROM AUTHOR]