Purpose: This study aimed to examine middle-distance runners’ acute physiological and biomechanical responses to 2 maximal aerobic speed tests: an intermittent test (5 × 1 mintest) and a continuous test (University of Montreal Track Test [UM-TT]). Methods: Twenty trained male middle-distance runners completed the UM-TT and 5 × 1 mintest on separate days. The rating of perceived exertion, peak heart rate, and maximal aerobic speed of both tests (VUM-TT and V5 ×1m) were analyzed. Heart-rate variability (time and frequency domain) and spatiotemporal parameters were measured preexercise and postexercise at 5, 30, and 60 minutes. Results: No differences were found between VUM-TT and V5×1m (18.4 [1.0] vs 18.5 [0.9] km·h−1, r = .88, P = .14). Peak heart rate was similar between tests (UM-TT: 188.6 [4] beats·min−1 vs 5 × 1 mintest: 189 [2] beats·min−1, r = .63, P = .23). Rating of perceived exertion was higher in UM-TT (UM-TT: 8.1 [0.5] vs 5 × 1 mintest: 7.6 [0.7], r = .47, P = .009). For both tests, heart-rate-variability parameters indicated higher sympathetic/lower parasympathetic activity compared with baseline at all time points (P < .001), without differences between tests. Stride frequency and duty factor increased postexercise (P < .05), with no changes in contact time and stride time (P > .05), without differences between the 2 tests. Conclusions: Both tests significantly changed heart-rate variability and biomechanical parameters, with no differences between the tests. As 5 × 1 mintest was perceived as less demanding, it may be a less taxing alternative for assessing maximal aerobic speed in middle-distance runners.