Glycerol has been used as a means to hyperhydate the body in attempts to offset deleterious effects of dehydration in addition to purported ergogenic effects on performance and thermoregulation. However, results of studies have been equivocal. Animal studies have reported serum glycerol saturations of 1.1 to 1.6 mmol/L associated with urinary glycerol excretion. When supplemental glycerol is lost through urination, the osmotic gradients associated with a given serum glycerol concentration that drive hyperhyration begin to diminish. Other than the animal studies of urinary glycerol excretion, little is known about similar responses in humans. PURPOSE: To identify the serum glycerol concentration coinciding with urinary glycerol excretion to determine if this may be a factor that may explain equivocal findings in glycerol hyperhydration studies. METHODS: Twelve healthy, trained male subjects completed five separate trials under resting conditions in a balanced predetermined sequence. During the trials, the subjects consumed a different glycerol dose (0.025, 0.05, 0.10, 0.15, or 0.20 g glycerol/kg LBM) of a 5% glycerol solution in order to determine at what serum glycerol concentration an increase in urine glycerol concentration becomes apparent. RESULTS: Based on regression analysis, serum glycerol concentrations 0.327 ± 0.190 mmol/L and a glycerol dose 0.032 ± 0.010 g glycerol/kg LBM would be associated with urinary glycerol excretion. There were significant (p < 0.001) linear relationships between peak serum glycerol response and time to reach peak serum glycerol response to ingested glycerol doses. CONCLUSIONS: This was the first study to identify the serum glycerol concentration associated with urinary glycerol excretion in humans. Since hyperhydration studies typically utilize a glycerol dose of 1.0 to 1.2 g/kg BW, urinary glycerol excretion becomes an issue in the maintenance of hyperhydration. Further research is warranted to examine maintenance doses of glycerol in hyperhydration applications in order to preserve serum glycerol concentrations required for sustained hyperhydation. This research was funded by grants from the UNM OGS and GPSA. Disclaimer: the views expressed are those of the authors and do not reflect the official policy or position of the USAF, the DoD, or the US Government.