The purpose of this study was to determine the effect of a continuous 110-km march with a 20-kg backpack load on plasma volume and intravascular protein content. Twenty-two healthy male volunteers, aged 19 to 20 years (mean, 19.4 years), physically conditioned for continuous strenuous exercise, with a mean (+/- SD) maximal oxygen consumption of 59.1 (+/- 7.9) ml/kg/min, participated in the study. The march was performed under ambient conditions of 17 to 32 degrees C dry temperature and 45 to 85% relative humidity. Venous blood samples were obtained before, during, and after the march. The average calculated oxygen consumption during the march was about 30% of maximal oxygen consumption. Mean body weight loss was 3.4% of the premarch weight, mean water ingestion was 14,250 ml, and mean urine volume was 2,687 ml. Relative changes of plasma volume and total content of plasma protein were calculated from hematocrit ratio and hemoglobin concentration. A significant reduction (-6.1 +/- 1.7%, mean +/- SE) in plasma volume and a minimal elevation in intravascular protein content (1.6 +/- 2.5%) were observed during the march. During the first 24 hours of recovery, plasma volume was further reduced (-8.4 +/- 1.8%), with a significant reduction in protein content (-6.6 +/- 1.8%), mainly albumin (-9.3 +/- 1.7%). During the second day of recovery, peak elevations in plasma volume (3.7 +/- 1.4%) and protein content (6.0 +/- 1.6%) were observed. The changes in protein content were related to elevations in albumin (3.7 +/- 1.3%) and globulin (10.7 +/- 3.2%) content. The elevated plasma volume and protein content were also maintained 96 hours after the end of the march. Although the changes in plasma volume during the march were associated with changes in serum albumin and globulin content, during the recovery period there was association only with the changes in serum globulin content. The possible mechanism of these findings is discussed.