The menstrual cycle is an important biological rhythm in women of reproductive age, characterised by rhythmic variation in hormone levels. Cyclical fluctuations in hormone profiles across the menstrual cycle are associated with alterations in metabolic control [1,2] with insulin sensitivity reduced during the luteal phase [2]. However, to date, research has yielded inconsistent results. In this study we firstly aimed to characterise the variation in insulin sensitivity and associated metabolites across the menstrual cycle. Secondly, we aimed to investigate the role of BMI, physical activity and cardiovascular fitness on variation in insulin sensitivity and associated metabolites across the menstrual cycle. Data from 1906 pre-menopausal women in NHANES cycles 1999-2006 were analysed. Participants in NHANES completed an at-home interview and physical examination. Menstrual cycle day was retrospectively assigned using questionnaire responses to “number of days since last period started”. All data for each participant are relevant to this single timepoint within their menstrual cycle. Rhythmic variation of plasma glucose, triglyceride, insulin, homeostatic model of insulin resistance (HOMA-IR) and adipose tissue insulin resistance index (ADIPO-IR) across the menstrual cycle were analysed using cosinor rhythmometry. Cosinor fits a cosine curve to data and calculates the MESOR (rhythm adjusted mean), amplitude (half the predictable variation within a cycle) and acrophase (time of highest value within a cycle). Participants were assigned low or high categories of BMI, physical activity and cardiorespiratory fitness and category membership included in cosinor models as covariates. Rhythmicity was demonstrated by a significant cosine fit for glucose (MESOR: 85.1 ± amplitude: 1.2 mmol/L; p= 0.014), but not triglyceride (87.7 ± 2.8 mg/dL; p= 0.369), insulin (9.8 ± 0.4 mmol/L; p= 0.470), HOMA-IR (2.1 ± 0.1 mmol/L; p= 0.461) or ADIPO-IR, (9.7 ± 0.6 mmol/L; p= 0.335). When covariates were included, rhythmicity was observed when adjusting for: 1. BMI: glucose (p< 0.001), triglyceride (p< 0.001), insulin (p< 0.001), HOMA-IR (p< 0.001) and ADIPO-IR (p< 0.001); 2. Physical activity: glucose (p 0.05). In conclusion, our study confirms previous reports showing insulin sensitivity undergoes small, yet statistically significant, rhythmic cycling across the menstrual cycle. This is the first study to demonstrate a modifying effect of BMI, physical activity and cardiorespiratory fitness on variation in insulin sensitivity and associated metabolites across the menstrual cycle. These findings demonstrate that menstrual cycle phase is an important consideration when assessing insulin sensitivity in clinical practice or research, especially in populations with high BMI or low cardiorespiratory fitness. Furthermore, this provides direction for investigation into the therapeutic benefit of targeting BMI and physical activity to mitigate disturbances in insulin sensitivity across the menstrual cycle.