Purpose Splenic contraction increases circulating hemoglobin (Hb) with advantages during hypoxia. As both hypoxia and exercise have been shown to be important separate triggers of splenic contraction we aimed to investigate if the spleen response to simulated high altitude (HA) is enhanced by superimposing exercise. Method Fourteen healthy volunteers (seven females) performed the following protocol in a normobaric environment sitting on an ergometer cycle: 20 min rest in normoxia; 20 min rest while breathing hypoxic gas simulating an altitude of 3500 m; 10 min exercise at an individually set intensity while breathing the hypoxic gas; 20 min rest in hypoxia; and finally 20 min rest in normoxia. Spleen measurements were collected by ultrasonic imaging and venous Hb measured at the end of each intervention. Result Mean ± SD baseline spleen volume during normoxic rest was 280 ± 107 mL, the volume was reduced by 22% during rest in hypoxia to 217 ± 92 mL (p < 0.001) and by 33% during exercise in hypoxia (189 mL; p < 0.001). Hb was 140.7 ± 7.0 g/L during normoxic rest and 141.3 ± 7.4 g/L during hypoxic rest (NS), but increased by 5.3% during hypoxic exercise (148.6 ± 6.3 g/L; p < 0.001). Spleen volume and Hb were stepwise changed back to baseline at cessation of exercise and return to normoxia. Conclusion Splenic contraction is induced by hypoxia and further enhanced by superimposing exercise, and reduced when exercise ceases, in a step-wise manner, showing that the tonic but partial contraction observed in long-term field expeditions to HA may occur also in the short term. This “graded response” may be beneficial during acclimatization to HA, to cope with moderate chronic hypoxia during rest while allowing additional enhancement of oxygen carrying capacity to overcome short bouts of extreme hypoxia caused by exercise.