In the present study, tissue pressure in the peritendinous area ventral to the human Achilles tendon was determined. The pressure was measured during rest and intermittent isometric calf muscle exercise at three torques (56, 112, and 168 Nm) 20, 40 and 50 mm proximal to the insertion of the tendon in 11 healthy, young individuals. In all experiments a linear significant decrease in pressure was obtained with increasing torque [e.g., at 40 mm: -0.4 +/- 0.3 mmHg (rest) to -135 +/- 12 mmHg (168 Nm)]. No significant differences were obtained among the three areas measured. On the basis of these observations, microdialysis was performed in the peritendinous region with a colloid osmotic active substance (Dextran 70, 0.1 g/ml) added to the perfusate with the aim of counteracting the negative tissue pressure. Dialysate volume was found to be fully restored (100 +/- 4%) during exercise. It is concluded that a marked negative tissue pressure is generated in the peritendinous space around the Achilles tendon during exercise in humans. Negative tissue pressure could lead to fluid shift and could be involved in the increase in blood flow previously noted in the peritendinous tissue during exercise (H. Langberg, J. Bülow, and M. Kjaer. Acta Physiol. Scand. 163: 149-153, 1998; H. Langberg, J. Bülow, and M. Kjaer. Clin. Physiol. 19: 89-93, 1999).