Background: Fetal Alcohol Spectrum Disorder (FASD) is a consequence of prenatal alcohol exposure (PAE) associated with a range of effects, including dysmorphic features, prenatal and/or postnatal growth problems, and neurodevelopmental difficulties. Despite advances in treatment methods, there are still gaps in knowledge that highlight the need for further research. The study investigates the effect of PAE on the autonomic system, including sex differences that may aid in early FASD diagnosis, which is essential for effective interventions. Methods: During gestational days 5 to 20, five pregnant female Wistar rats were orally administered either glucose or ethanol. After 22 days, 26 offspring were born and kept with their mothers for 21 days before being isolated. Electrocardiographic recordings were taken on the 29th and 64th day. Heart rate variability (HRV) parameters were collected, including heart rate (HR), standard deviation (SD), standard deviation of normal-to-normal intervals (SDNN), and the root mean square of successive differences between normal heartbeats (RMSSD). Additionally, a biochemical analysis of basic serum parameters was performed on day 68 of the study. Results: The study found that PAE had a significant impact on HRV. While electrolyte homeostasis remained mostly unaffected, sex differences were observed across various parameters in both control and PAE groups, highlighting the sex-specific effects of PAE. Specifically, the PAE group had lower mean heart rates, particularly among females, and higher SDNN and RMSSD values. Additionally, there was a shift towards parasympathetic activity and a reduction in heart rate entropy in the PAE group. Biochemical changes induced by PAE were also observed, including elevated levels of alanine transaminase (ALT) and aspartate aminotransferase (AST), especially in males, increased creatinine concentration in females, and alterations in lipid metabolism. Conclusions: PAE negatively affects the development of the autonomic nervous system, resulting in decreased heart rate and altered sympathetic activity. PAE also induces cardiovascular abnormalities with sex-specific effects, highlighting a relationship between PAE consequences and sex. Elevated liver enzymes in the PAE group may indicate direct toxic effects, while increased creatinine levels, particularly in females, may suggest an influence on nephrogenesis and vascular function. The reduced potassium content may be linked to hypothalamus–pituitary–adrenal axis overactivity.