The objective of the study was to determine if nuclear magnetic resonance (NMR) spectral features of ovarian follicular fluid were correlated with the physiological status of follicles so that we could assess the feasibility of using NMR spectroscopy during assisted reproduction therapy. Thirty-five sexually mature, nullparious heifers were monitored by transrectal ultrasonography to assess their follicle wave status during the oestrous cycle. Ovariectomies were performed on Day 3 of wave 1 (D3W1, n = 10), Day 6 of wave 1 (D6W1, n = 9), Day 1 of wave 2 (D1W2, n = 9), or in the immediate preovulatory period of at least 17 days after ovulation (D≥17, n = 9). Follicle status was determined to be dominant or subordinate. Follicular fluid was extracted from the follicles and NMR spectra were collected. Principal components were extracted from ratios of line amplitudes and tested for effects of follicle status (dominant v. subordinate) and cycle time point (D1W3, D1W6, D1W2 and D≥17) using multivariate analysis of variance. For most line ratio combinations, main effects of status, time point and their interaction were found (P < 0.05). We concluded that NMR spectra may be used for the determination of ovarian follicle physiological status.