The physiological and metabolic effects of prophylactic treatment with osmolytes were investigated using twenty-four 2.5-yr-old Bos indicus steers. Animals were allocated to 1 of 4 treatment groups: 1) control, feed and water deprived for 48 h (n = 6); 2) transported, transported for 48 h (n = 6); 3) glycerol, dosed with glycerol (2 g/kg of BW) and then transported for 48 h (n = 6); and 4) betaine, dosed with betaine (0.25 g/kg of BW) then transported for 48 h (n = 6). Body water, electrolytes, blood pH and gases, plasma lactate, glucose, albumin, total protein, anion gap, strong ion difference, total weak acids, and BW were determined at the conclusion of 24 and 48 h of transportation. The glycerol group had greater body water volumes than the control (P = 0.05) and transported (P = 0.02) groups. The glycerol, transported, and betaine groups had lower (P = 0.02) plasma Mg concentrations than the control group at 24 h, whereas the glycerol group maintained lower (P = 0.04) plasma concentrations of Ca than the control group. The betaine group had lower (P = 0.04) hematocrit than the control group at 24 and 48 h. Plasma bicarbonate and pCO2 were 13 and 17% greater (P = 0.01 and 0.04, respectively) in the glycerol group at 24 h compared with control and transported groups. However, the ratio of [HCO3]/[CO2] in the glycerol group did not differ from the other groups and thereby maintained pH. The glycerol group maintained a 30% greater (P < 0.001) plasma concentration of glucose than the control group, and 14% greater (P = 0.05) than the transported and betaine groups. In contrast, betaine had little effect on increasing blood glucose compared with glycerol. Glycerol-linked hyperhydration at 24 h may not only help to conserve water loss during long distance transportation, but the increased blood glucose may have an important protein-sparing effect due, in part, to greater insulin concentrations inhibiting the breakdown of muscle proteins, thus, countering the amino-acid mobilizing effect of cortisol after 24 h. Therefore, the osmolyte glycerol shows promise as a prophylactic treatment for attenuating the effects of long distance transportation by maintaining body water, decreasing the energy deficit, and preserving health and muscle quality.