The objectives of the present study were to investigate the effects of Aquatraining of horses (aqua-treadmill exercise; treadmill manufactured by Equitech – L.u.S. Equipment, Warendorf, Germany) on selected blood parameters [lactic acid concentration (mmol/l), haemoglobin content (g/l)] and on heart-rate variability (HRV) [heart rate (beats per min; b.p.m.), standard deviation of all NN-intervals (SDNN; ms), normalized power of the low and high frequency band (LFnorm, Hfnorm; au), % recurrence, % determinism and ratiocorr]. Seven horses performed six exercise tests with different work loads (walking (x=1.56 ± 0.08 m/s) and trotting (x=2.9 ± 0.13 m/s); dry, water above the carpus and water above the elbow). The standardized test-protocol was: 5 min warm-up at walk while the water was pumped in, followed by the 20-min exercise period at walk or trot, followed by a 5-min walk while pumping out the water. Blood samples were taken prior to each test at rest in the stable, as well as exactly 5 min after the end of the 20-min exercise period. Electrocardiograms were recorded during rest and the 20-min exercise period. Compared to rest, neither the chosen velocities, the two water levels, nor the dry tests led to a significant increase of the lactic acid concentration in any horse. The haemoglobin content showed a significant increase as a result of exercise. Significant differences could be found between the heart rates at rest and the six exercise tests and between the mean of the levels `walking' and the mean of the levels `trotting'. An exercise-induced change of HRV was characterized by a decreasing SDNN, a significantly higher LFnorm (sympathetic influence) combined with a significantly lower HFnorm power (parasympathetic activity) and a rising degree of order (significantly higher % determinism and nearly unchanged % recurrence) and stability (significantly rising ratiocorr) of the recurrence plot. In conclusion, the used training-protocol for aqua-treadmill exercises only represents a medium-sized aerobic work load for horses, but the different levels of burden were indicated especially by changes in HRV.