The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses. We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively. We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period. The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals. The contaminated diet suppressed secondary antibody responses, but only in the morning. The contaminated diet also altered CYP1A, but not vitellogenesis. In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction. Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females. This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.