Background and objectives Type 2 Diabetes mellitus (T2DM) is a heterogeneous group of metabolic disorders with variable degrees of insulin resistance and altered glucose metabolism. Increased attention in studying the role of gonadal hormones in diabetes is not only due to their relation to insulin sensitivity, and glucose tolerance but also to the gender-specific nature of the prevalence of various diabetic complications. The cyclical change in the hormone level in females will make it necessary to consider the menstrual cycle while analyzing the risk factors for diabetes. Hence, the role of gender and menstrual cycle in T2DM are analyzed here using the simple non-invasive cardiovascular risk indices like heart rate variability (HRV), QT interval corrected for heart rate (QTc), and JT interval. Materials and methods In this analytical study, T2DM patients in the age group of 18-45 years with less than five years duration from diagnosis and taking not more than two anti-hyperglycemic drugs were included. Time and frequency domains of HRV analysis, QTc, and JT intervals were compared with age and BMI matched control group. The comparison of these parameters was also made between two genders in the diabetic group and they were analyzed across different phases of the menstrual cycle in female diabetic patients when physiological variation in the gonadal hormones occurred as a natural phenomenon. Results HRV parameters were reduced and the QTc and JT intervals were prolonged in diabetic patients of both genders. Reduction in low-frequency (LF) band power and high-frequency (HF) band power of HRV analysis in diabetic females were statistically significant in the luteal phase of the menstrual cycle in comparison with age and BMI-matched healthy controls. There was no significant difference in the HRV parameters, QTc, and JT interval between the male and female diabetic groups. HF band power is significantly reduced in the menstrual phase and relatively higher in the follicular phase when compared to the luteal phase among female diabetic patients. Conclusion The reduced sympathetic and parasympathetic activity were observed in diabetic patients of both genders and they were significant in the luteal phase of diabetic females compared to the healthy control group. Vagal activity is relatively higher in the follicular phase of the menstrual cycle in female diabetic patients.