Background: Although the initial anaerobic component of exercise adaptation is unavoidable, no specific functional test is available for use in routine non-sporting practice to evaluate it. Objective: To assess the bioenergetic and biomechanical properties of the Short and Fast Step Test (SFST), which consists of walking up and down a step as many times as possible in 1 minute and to analyse its ability to explore the initial anaerobic component of effort in comparison to a reference self-paced step test. Methods: Overall, 31 healthy subjects (19 women; mean [SD] age, 32.4 [10.2] years) completed a test–retest of a self-paced step test and the SFST, with pre- and post-test measurement of blood lactate concentration and continuous recording of VO2 and modelling of excess post-exercise oxygen consumption (EPOC), near-infrared spectroscopy (NIRS) of the quadriceps and mechanical power (estimated by the number of steps climbed and 3-D motion analysis). Results: Both step tests were well tolerated. The reliability of the bioenergetics parameters, number of raised steps, mechanical power and NIRS tissue saturation index was good. Indirect mechanical power (estimated from number of steps) was correlated with direct power (computed from the centre of mass). Lactate accumulation was significantly increased during exercise with only the SFST (mean [SD] increase, 3.86 [3.26] mmol L−1 from resting values, P < 0.05). EPOC was higher with the SFST than the self-paced step test (P < 0.05). Only the SFST showed significant correlations between number of steps climbed and EPOC (r = 0.84; P < 0.001) and decreased tissue saturation index (NIRS) and EPOC area (r = −0.39; P < 0.05). Conclusions: SFST is feasible, well tolerated, reliable and responsive to explore a brief exercise involving anaerobic metabolism at submaximal intensity. The number of steps taken in 1 minute seems a suitable parameter for practical application.