Due to climate change, water level fluctuations (WLFs) in shallow lakes are expected to increase in the future. Such fluctuations significantly affect the growth and reproduction of submerged macrophytes, but the role of WLF on plant physiological mechanisms is far from clear. Our aims are to examine the effects of WLF on the submerged macrophyte Vallisneria spinulosa by testing changes in (1) chloroplast pigments, (2) the response of antioxidant enzymes, (3) the concentrations of carbon (C), nitrogen (N) and phosphorus (P) and (4) plant stoichiometry (ratios of C/N, C/P and N/P). In outdoor mesocosms, plants were subjected to two amplitudes and two frequencies of WLF, with stable water level as a control. Generally, compared with the control, the fluctuation treatments showed higher chlorophyll a and b concentrations. Increased amplitude and frequency promoted peroxidase (POD) activity in V. spinulosa, indicating a response to oxidative stress, but superoxide dismutase (SOD) remained unchanged. Moreover, compared with the control, WLF generally increased leaf P and reversely affected the leaf C/P and N/P ratios of plants. The C and N contents in the plants were relatively stable following WLF, but with increasing WLF amplitude, P absorption (i.e. leaf P content) by V. spinulosa was promoted. Our results indicate that the ability of V. spinulosa to successfully grow in floodplains may be related to the physiological adaptive strategies according to the alterations in WLF. Combined effects of several hydrological factors on shallow lake should be further studied in order to maintain the health and sustainability of shallow lake systems.