The present study aimed to evaluate the effects of a highly-demanding match and a lowly-demanding match on pre-competitive heart rate variability (HRV) and anxiety in semi-professional female soccer athletes. A total of 14 players, with a mean age of 23.78 (4.93), from the Caceres Women Football Club of the Spanish Second National Division participated in our study. They were evaluated in two microcycles which correspond to a highly- and a lowly-demanding matches. For each microcycle a baseline and a pre-competitive measures were collected. Results indicated that HRV was significantly reduced before a highly demanding match whereas a lowly-demanding match did not lead to any change. Significant differences in HRV and cognitive anxiety were observed when compared the highly and the lowly demanding matches, which means an increase in the anxiety levels before the highly-demanding match. HRV could be an indicator of precompetitive anxiety in semi-professional female soccer players. This could be used by coaches or physical trainers as a tool to examine the precompetitive anxiety in athletes.