Glycerol is prohibited as a masking agent by the World Anti-Doping Agency and a urinary threshold has recently been recommended. However, little is known about urinary glycerol excretion after exercise, when (1) exogenous glycerol is metabolized increasingly and (2) endogenous glycerol levels are elevated. The purpose of the placebo-controlled cross-over study was to determine the effects of pre-exercise glycerol administration on glycerol metabolism, urinary excretion, and selected blood parameters. After administration of glycerol (G; 1.0 g/kg body weight (BW) + 25 ml fluid/kg BW) or placebo (P; 25 ml fluid/kg), 14 cyclists exercised 90 min at 60% VO2max. Samples were taken at 0 h (before administration), 2.5 h (before exercise), 4 h (after exercise) and 6.5 h and additional urine samples were collected until 24 h. Exercise increased endogenous plasma glycerol (0.51 ± 0.21 mmol/l) but peak concentrations were much higher in G (2.5 h: 15.6 ± 7.8 mmol/l). Urinary glycerol increased rapidly (58,428 ± 71,084 µg/ml after 2.5 h) and was significantly higher than in P until 13.6 ± 0.9 h (p < 0.01). In comparison with placebo administration, G caused significantly greater changes in plasma volume and haemoglobin concentrations after 2.5 h. BW and urine production were significantly different between P and G after 2.5 h and post-exercise. Despite exercise-induced increases in endogenous glycerol in the control group, urinary excretion remained well below the previously recommended threshold. In addition, exercise-related glycerol degradation did not appear to negatively affect the detection of exogenously administered glycerol. Copyright © 2013 John Wiley & Sons, Ltd.