(1) Research on heart rate variability has increased in recent years and the temperature has not been controlled in some studies assessing repeated measurements. This study aimed to analyze how heart rate variability may change based on environmental temperature during measurement depending on parasympathetic and sympathetic activity variations. (2) A total of 22 volunteers participated in this study divided into an experimental ( = 12) and control group ( = 10). Each participant was assessed randomly under two different environmental conditions for the experimental group (19 °C and 35 °C) and two identical environmental conditions for the control group (19 °C). During the procedure, heart rate variability measurements were carried out for 10 min. (3) Significantly changes were observed for time and frequency domains as well as Poincaré plot variables after heat exposure ( < 0.05). These findings were not observed in the control group, whose conditions between measurements did not change. (4) The reduction of heart rate variability due to exposure to hot conditions appears to be produced mostly by a parasympathetic withdrawal rather than a sympathetic activation. Therefore, if consecutive measurements have to be carried out, these should always be done under the same temperature conditions.