Musical interpretation produces psychophysiological medium to long term alterations in the instrumentalist, with lack of knowledge about acute changes associated to conditional fatigue (1, 2). On the other hand, the coordination between rhythm and biological series like heart beating or breathing are a matter of recent research (3). Pulmonary and cardiac synchronic modulation improves after playing wind instruments, reflecting an increase in vagal control and a concomitant augment in Heart Rate Variability (HRV) (4, 5). The influence of emotion on the autonomic modulation, according to the polyvagal theories (4) is also accepted. However, there is little research concerning the influence of mediating factors like the Perceived Difficulty (PD). The present study aims to analyze time course changes in HRV following one hour of performance, looking for acute fatigue symptoms. It analyzes two levels of PD, minimizing psychological stress.8 male wind instrument musicians (29.13±7.33 years; 69.36±10.31 kg) ranked a list of well-known performances in terms of PD. Later on, in two normal rehearsals with no audience (alternate days), musicians performed a mild performance (M), and the most difficult one (D). They were previously advised about the performances, so they could increase their practice. After 10 min of warm up, they played twice for 20 min, with 5 min of rest. HR was recorded (Polar 810) in a sitting position, including 20 min previous to the performance and post-performance registers. The last 500 beats within each 20 min in the four sample conditions (pre; per1; per2 & post performance) were retained for further analysis with Kubios (software 2.1). A Repeated Measures ANOVA, followed by Bonferroni post-hoc, was conducted to search differences in HRV due to time playing. Differences in HRV time course regarding PD were also examined. Due to the non-stationarity of the cardiac signal, RRi, RMSSD (Root Mean Square of Successive Differences) and SD1, SD2, and SD1/SD2 ratio (Poincare Plot indexes) were chosen. Univariate contrast showed significant changes in RRi and SD1/SD2 (p<0.005), and SD2 (p<0.05) regarding time curse, with no differences in lnRMSSD and lnSD1. Paired comparison exhibited interesting differences in time course according to PD (Figure 1; Bonferroni) RRi and SD2 (HRV indexes related to sympathetic system) describe an inverse curve which unexpectedly started to come back to baseline conditions during the second part of the performance. After a first increase in the adrenergic response (Per1),bigger for D, the curve displayed a trend to a vagal reactivation in Per2 (i.e. higher SD1 and lnRMSSD, higher SD2) although this is not significant. Thus, one hour playing is not a strenuous situation, even in performances perceived as difficult. The study confirms beneficial effects on autonomic functioning, at least when psychological stress is under control.