The present study analyzes the testosterone (T), cortisol (C) and emotional response in competitive interactions between dyads, as well as the relationship between basal T and the emotional response. Seventy-two men and women (36 dyads) participated in same-sex dyads in a face-to-face laboratory competition, and thirty-two men and women (16 dyads) carried out the same task in a non-competitive condition. Salivary samples (5 ml of saliva, plastic vials) were provided at three time points (baseline, task, and post-task), and subsequently T (pg/ml) and C (nmol/L) concentrations were measured using ELISA method. Participants completed self-reported measures of emotional valence, emotional arousal and perceived dominance by means of the Self-Assessment Manikin (SAM), at three time points (pre-task, task, and post-task). Two-level crossed Multilevel Models (MLM) showed a participants' stability in C (Mean ± SEM: baseline: 3.84 ± 0.28, task: 2.92 ± 0.28 and post-task: 2.62 ± 0.3), emotional valence (pre-task: 4 ± 0.06, task: 3.66 ± 0.1 and post-task: 3.84 ± 0.09), arousal (pre-task: 3.29 ± 0.09, task: 3.83 ± 0.09 and post-task: 3.38 ± 0.1) and dominance (pre-task: 3.28 ± 0.08, task: 3.4 ± 0.1 and post-task: 3.44 ± 0.09) values, which in the case of emotional valence and dominance was modulated by time-point, outcome and sex. Furthermore, analyses revealed that opponents' C, arousal and dominance values at one time-point influenced participants' values at the following time-point modulated by outcome, sex and time-point. Moreover, MLM indicated that in loser men, individuals higher in basal T (126.31 ± 6.4) displayed higher negative emotional valence after the defeat (post-task: 3.6 ± 0.21), while in women basal T (99.78 ± 12.6) was not significantly related to post competition emotional valence. These findings reinforce the importance of studying the relationship between hormonal and psychological changes in dyadic competition, and confirm that men and women differ in their psychophysiological responses to competition.