Abstract Direct methanol fuel cell (DMFC) durability tests were conducted in three different operational modes: continuous operation with constant load (LT1), on–off operation with constant load (LT2) and on–off operation with variable load (LT3). Porous carbon nanofiber (CNF) anode layers were employed in three sets of single passive DMFCs; each membrane electrode assembly (MEA) was run continuously in durability testing for 3000 h. The objective of this study is to investigate the degradation mechanisms in an MEA with a porous CNF anode layer under different modes of operation. The polarization curves of single passive DMFCs before and after durability tests were compared. The degradation of DMFC performance under the cyclic LT1 mode was much more severe than that of LT2 and LT3 operation. The loss of maximum power density after degradation tests was 49.5%, 28.4% and 43.7% for LT1, LT2 and LT3, respectively. TEM, SEM and EDS mapping were used to investigate the causes of degradation. The lower power loss for LT2 was mainly attributed to the reversible degradation caused by poor water discharge, which thus reduced the air supply. Catalyst agglomeration was especially observed in LT1 and LT3 and is related to carbon corrosion due to possible fuel starvation. The loss of active catalyst area was a major cause of performance degradation in LT1 and LT3. In addition to this, the dissolution and migration of Ru catalyst from the anode to cathode was identified and correlated with degraded cell performance. In the DMFC, the carbon nanofiber anode catalyst support exhibited higher performance stability with less catalyst agglomeration than the cathode catalyst support, carbon black. This study helps understand and elucidate the failure mechanism of MEAs, which could thus help to increase the lifetime of DMFCs.