OBJECTIVE: To test the operational viability of and validate the 4-second exercise test (4sET) protocol in the orthostatic position (ORTHO). METHODS: The ORTHO protocol, an alternative to the conventional protocol (CYCLO), was used. The ORTHO protocol consists of performing sudden exercise in the orthostatic position – accelerated stationary walking (alternate upward flexion of the thighs) – from the fourth to the eighth second of a 12-second maximum inspiratory apnea, instead of rapid cycling without load. The adimensional cardiac vagal index (CVI) was calculated using the ratio between the longest RR interval (RRB) – the one immediately before, or the first during exercise – and the shortest RR interval during exercise – usually the last (RRC) – measured on electrocardiographic tracings at a 10-ms resolution. Forty-seven individuals (40±17 years, 169±9 cm, 72±14 kg) of both sexes, healthy or unhealthy, randomly underwent 3 consecutive repetitions of the 2 protocols, the first being performed only to acquaint patients with the procedure. RESULTS: Although differences in the CVI were found in both protocols (1.48±0.04 vs 1.42±0.04; P<0.001), no physiological relevance was observed. In 5 (11%) cases, a clinically significant difference between the ORTHO and CYCLO protocols was observed for CVI. The results of RRB, RRC, and CVI in the 2 protocols were strongly correlated, being 0.84, 0.85, and 0.93, respectively (P<0.001). CONCLUSION: The 4sET performed in the orthostatic position proved to be a valid option for assessing the vagal cardiac tonus in laboratories lacking a cycloergometer, without jeopardizing clinical interpretation. In addition to simplicity and applicability, the procedure also provides low operational costs.