The specifics of short-term physical exercise are similar to the immediate reaction demands placed on police officers. Identifying the physiological predisposition to short-term high-intensity exercise in male law enforcement officers will assist in understanding their metabolism and make a significant contribution to a much more personal and individualized workout program. This will improve physical fitness of individual officers, improving their preparedness for such times of emergency. This cross-sectional study was conducted to investigate the responses of hematological (erythrocytes, hemoglobin, hematocrit, leucocytes, monocytes, neutrophils, lymphocytes), hormonal (testosterone, cortisol, melatonin), biochemical (glucose, uric-acid, lactate, creatine-phosphokinase) data to short-term maximal exercise in male police officers ( n = 20). Blood samples were collected before- and after- the running-based anaerobic sprint test (RAST), and biological values were corrected for fluid shifts. Data were mean ± standard deviation of differences (= after minus before RAST). After the RAST, values of cortisol, lactate, neutrophils, lymphocytes, and monocytes increased significantly by 7.01 ± 37.36 mmol/l, 7.55 ± 1.67 mmol/l, 0.17 ± 0.26 10³/µl, 0.61 ± 0.28 10³/µl, and 0.10 ± 0.13 10³/µl, respectively. After the RAST, values of melatonin, uric-acid, creatine-phosphokinase, hemoglobin, and hematocrit decreased significantly by −13.24 ± 4.60 pg/ml, −13.28 ± 14.35 µmol/l, −10.23 ± 10.13 IU/l, −2.01 ± 0.81 g/dl, and −4.46 ± 0.59%, respectively. Biological data of male police officers were affected by sprint test. Understanding changes in biological data following short-term maximal exercise can further assist in a better understanding of anaerobic metabolism, which will be helpful to find available methods for coaches to quantify training loads.