A major goal of biomedicine is to understand the function of every gene in the human genome. Null mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. To date, comprehensive analysis of genes knocked out in humans has been limited by the fact that null mutations are infrequent in the general population and so, observing an individual homozygous null for a given gene is exceedingly rare. However, consanguineous unions are more likely to result in offspring who carry homozygous null mutations. In Pakistan, consanguinity rates are notably high. Here, we sequenced the protein-coding regions of 7,078 adult participants living in Pakistan and performed phenotypic analysis to identify homozygous null individuals and to understand consequences of complete gene disruption in humans. We enumerated 36,850 rare (<1 % minor allele frequency) null mutations. These homozygous null mutations led to complete inactivation of 961 genes in at least one participant. Homozygosity for null mutations at APOC3 was associated with absent plasma apolipoprotein C-III levels; at PLAG27, with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; and at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations. After physiologic challenge with oral fat, APOC3 knockouts displayed marked blunting of the usual post-prandial rise in plasma triglycerides compared to wild-type family members. These observations provide a roadmap to understand the consequences of complete disruption of a large fraction of genes in the human genome.