In this study, we describe the colorimetric detection of lactate concentration in real human sweat samples, as an indication of the performance of especially athletes, on a microfluidic device fabricated on a microfibrous nonwoven substrate comprising microfluidic channels and detection reservoirs. A narrow group (10 healthy individuals: 5 women and 5 men) within age range of 20-25 was used. The optimization and real sweat studies demonstrated that color formation on the biosensor surface is influenced not only by the enzyme, ABTS, gelatin ratios but also by many parameters such as storage conditions. Usage of real sweat samples showed that simulated samples only have limited similarity with the real samples and their contents can have a significant impact on the biosensor, in our case on color formation. To understand the response better, larger group of human subjects should be examined in detail in terms of effect of sweat properties to biosensor signal.