As nanoparticles could form aggregates in biological systems, the dynamics of their dispersity drives the temporal effect of nanoparticles in vitro. To test this hypothesis, the fumed silica nanoparticles (SiNPs) that have primary sizes of 7-14 nm and form aggregates in culture medium were selected for toxicity study in human lung A549 cells. The dispersity of SiNPs was analyzed by dynamic light scattering and transmission of electron microscopy. Cytotoxicity assays including mitochondrial activity, intracellular level of reactive oxygen species (ROS), and membrane damage together with the ¹H-NMR-based extracellular metabonomic assay were conducted to determine the temporal dose-effect relationship of SiNPs. In cell culture medium, SiNPs dispersed well initially at 25-100 μg/ml; however, they sedimented rapidly in a concentration-dependent manner. SiNPs caused a dose-dependent increase of intracellular ROS and cell membrane damage at 4 h and a loss of cell viability after 48 h. SiNPs also induced an elevation of extracellular glucose, lactate, phenylalanine, histidine, and tyrosine levels in a time- and concentration-dependent manner. The dose-effect patterns at 4 h were different from that at 12 and 24 h as assessed by both cytotoxicity and metabonomic assays. Both fitted better with polynomial regression than linear regression, implying multimode action of SiNPs at different concentrations. The early NP-cell interaction and the late sedimentation could be attributable to the temporal effects of SiNPs. The extracellular ¹H-NMR-based metabonomics demonstrated a potential as a robust nondestructive tool for monitoring the temporal effect of NPs that tend to aggregate in nature.