The objective of the present study was to evaluate the effect of intense training in endurance athletes on the behaviour of cholinesterases (ChE) after fatigue conditions and its relationship with other internal load markers. 18 male athletes specialized in endurance events participated. ChEs and two index of heart rate variability were evaluated at three different moments, before the study protocol (BASAL), 15 minutes after (FINAL) and the day after finishing the training (24H). A one-way ANOVA with post-hoc Tukey HSD was used to compare means. Significant differences were found in the variables analysed (p < .001), with very large effect sizes (d > 0.9) between BASAL, FINAL and 24H and moderate correlations between ChE and LnRMSSD and SS (p < .001). The behaviour of the ChEs showed a significant change (p < .001) after exercise and relationship with other internal training load indicators. Our results indicate that ChEs are related to fatigue in the studied athletes and may be a measure for training load determination.