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Description: The existence of ovulatory cycle shifts in women’s mate preferences has been discussed controversially. There is evidence that naturally cycling women in their fertile phase, compared to their luteal phase, evaluate specific behavioral cues in men as more attractive for sexual relationships. However, recent research has cast doubt on these findings. We addressed this debate in a large, pre-registered within-subject study including salivary hormone measures and luteinizing hormone tests. One-hundred-fifty-seven female participants rated natural videos of 70 men in dyadic intersexual interactions on sexual and long-term attractiveness. Multilevel comparisons across two ovulatory cycles indicated that women’s mate preferences for men’s behaviors did not shift across the cycle, neither for competitive, nor for courtship behavior. Hormone levels and relationship status did not affect these results. Hormonal mechanisms and implications for estrus theories are discussed.