Natural and human activities can result in both high temporal and spatial variability in water temperature. Rapid temperature changes have the potential to dramatically affect physiological processes in aquatic organisms and, due to their limited mobility, fish early life stages are particularly vulnerable to ambient temperature fluctuations. In this study, we examined how the magnitude and frequency of temperature fluctuations affect survival, growth, development, expression of thermoresponsive genes, and gonadal differentiation in fathead minnows, Pimephales promelas. We exposed individuals (0 to 4 days post fertilization) of known genotypic sex to fluctuations of Δ4 °C over 12-h, Δ8 °C over 12- and 24-h, and three stable temperatures (21, 25, and 29 °C) for up to 45 d. Expression of hsp70 in fish exposed to the highest-magnitude, highest-frequency fluctuating treatment cycled in concert with temperature and was upregulated initially during exposure, and may have contributed to temperature fluctuations having little effect on time to and size at hatching (whole-organism responses). This treatment also caused fish to undergo nondirectional sex reversal. These results indicate that hsp70 may be involved in mediating thermal stress from subdaily temperature fluctuations and that sex determination in fathead minnows can be influenced by cycling temperatures.