Dynamic strain ageing (DSA) in an austenitic Ni-based alloy was investigated by symmetric/asymmetric low cycle fatigue tests with different strain amplitudes at 350 °C. A quantitative analysis of DSA activity was provided in terms of the characteristic parameters (DSA transient and serration length), and associated microstructural features were explored. Particular attention was paid to the comparison in the DSA domain between Ni- and Fe-based austenite with different strain ratios. Results showed that DSA existed throughout life and led to the continuous cyclic hardening in austenitic Ni-based alloy. The DSA activity first decreased and remained almost constant under symmetric loadings. A gradual increase in DSA activity was observed for the asymmetric loadings before the final failure. In addition, a physical model was presented to explore the DSA mechanisms of austenitic Ni-based alloys in different temperature ranges. Specifically, at 350 °C, DSA is caused by C diffusion in austenite, while at higher temperatures, it may be caused by Cr diffusion. Moreover, for a given mechanism, the DSA occurred at a higher temperature range in Ni-based alloy than in Fe-based alloy.