Hydrogen isotope values (δD) of sedimentary terrestrial leaf wax such as n-alkanes or n-acids have been used to map and understand past changes in rainfall amount in the tropics because δD of precipitation is commonly assumed as the first order controlling factor of leaf wax δD. Plant functional types and their photosynthetic pathways can also affect leaf wax δD but these biological effects are rarely taken into account in paleo studies relying on this rainfall proxy. To investigate how biological effects may influence δD values we here present a 37,000-year old record of δD and stable carbon isotopes (δ13C) measured on four n-alkanes (n-C27, n-C29, n-C31, n-C33) from a marine sediment core collected off the Zambezi River mouth. Our paleo δ13C records suggest that each individual n-alkanes had different C3/C4 proportional contributions. n-C29 was mostly derived from a C3 dicots (trees, shrubs and forbs) dominant vegetation throughout the entire record. In contrast, the longer chain n-C33 and n-C31 were mostly contributed by C4 grasses during the Glacial period but shifted to a mixture of C4 grasses and C3 dicots during the Holocene. Strong correlations between δD and δ13C values of n-C33 (correlation coefficient R2 = 0.75, n = 58) and n-C31 (R2 = 0.48, n = 58) suggest that their δD values were strongly influenced by changes in the relative contributions of C3/C4 plant types in contrast to n-C29 (R2 = 0.07, n = 58). Within regions with variable C3/C4 input, we conclude that δD values of n-C29 are the most reliable and unbiased indicator for past changes in rainfall, and that δD and δ13C values of n-C31 and n-C33 are sensitive to C3/C4 vegetation changes. Our results demonstrate that a robust interpretation of palaeohydrological data using n-alkane δD requires additional knowledge of regional vegetation changes from which n-alkanes are synthesized, and that the combination of δD and δ13C values of multiple n-alkanes can help to differentiate biological effects from those related to the hydrological cycle.