The aim of the study was to compare the oxygen uptake (V˙O2 ), heart rate (HR), rated perceived exertion (RPE), and blood-lactate concentration (BLa) in a conventional anaerobic-threshold test at constant speed intervals with running at the same mean speeds, but with speed variations similar to a football match. Nine male football players completed two test days with two treadmill running tests: a submaximal test followed by a maximal test. The submaximal tests (tests A and B) comprised five stages of five minutes each, with between 8–16 km·h−1 mean speeds. The speed was constant for test A, whereas in test B, the speed during the stages varied every 15 seconds. Mean values of V˙O2 , HR, BLa, and RPE for the legs (RPElegs) and ventilation (RPEvent) were determined for each stage. No significant differences between tests A and B were found for V ˙O2 , HR, and RPElegs. The BLa was significantly higher for test B at mean speeds of 10, 14, and 16 km·h−1. RPEvent did not differ between tests for any work intensity, except for a higher rating for test A at 8 km·h−1. The equal HR and V ˙O2 responses for the anaerobic-threshold tests with constant and varying speeds suggests that male football players’ aerobic energy expenditure during match could be estimated based on HR recordings. The significant difference in BLa between constant and varying speeds indicates the need to use anaerobic-threshold tests with varying speeds in football.