Purpose: This study analyzed the physiological response during Yo-Yo Intermittent Recovery Level 1 (YYIR1) test and re-test by in-field ergospirometry and time-series analyses of respiratory parameters. Methods: Ten moderately trained males (23.4 ± 2.01 years, VO[subscript 2peak]= 56.81 ± 10.75 mL·kg[superscript -1]·min[superscript -1]) completed three running trials including two separate YYIR1 tests and an independent maximal performance running test with time-series analyses of gas exchange parameters. Physiological response was assessed during all tests by determination of blood lactate levels (including calculation of individual lactate threshold), heart rate, oxygen consumption and respiratory exchange ratio (RER). Results: Modeling of YYIR1 test mean VO[subscript 2] uptake kinetics over all participants revealed that VO[subscript 2] increased rapidly after the individual lactate threshold (11.49 ± 0.66 km·h[superscript -1] at 3.83 ± 0.42 mmol·L[superscript -1]) was reached with ~95% VO[subscript 2peak] at ~50% of the test duration (test, VO[subscript 2][superscript 50%]= 95.17 ± 8.74% of VO[subscript 2peak]; re-test, VO[subscript 2][superscript 50%]= 96.78 ± 7.04% of VO[subscript 2peak]). However, and despite identical YYIR1 test performance (1568 ± 364.6 m vs. 1568 ± 449.7 m, CV = 4.59%), mean VO[subscript 2peak] during YYIR1 test was 8.81 ± 5.6% higher than YYIR1 re-test (p = 0.027). Importantly, correlation of VO[subscript 2peak] with YYIR1 test performance was weak (R[superscript 2] = 0.28, p = 0.115). Conclusions: We conclude that the YYIR1 test should not be used to estimate VO[subscript 2peak]. Further studies on direct determination of gas exchange parameters during different YYI test variants are warranted.