Analysis of ammonium in human sweat during physical strain using a portable sensor device and enzymatic measurements are compared. The portable device is based on a screen‐printed electrolyte sensor connected with an evaluation board for data acquisition and transfer. During performance tests with artificial sweat, the sensor shows a low detection limit of 0.3 mM. Thus, the typical concentration range of ammonium in sweat lies within the working range of the sensor. We also demonstrate the necessity of the comparison measurement for verification of the correct performance of screen‐printed sensors and show how other components of human sweat influence the measured potential. Regarding the change in ammonium concentration during different levels of work out intensity, we point out a different behavior of what is expected physiologically. With this the importance of consideration of several parameters during sweating, like the sweat rate and other ingredients in human sweat, was demonstrated for correct determination of the ammonium concentration.